Soil and root populations of fluorescent Pseudomonas spp. associated with seedlings and field-grown plants are affected by sorghum genotype

Sorghum [Sorghum bicolor (L.) Moench] is valued for bioenergy, feed and food. Potential of sorghum genotypes to support differing populations of root- and soil-associated fluorescent Pseudomonas spp. or Fusarium spp., in two soils, was assessed. Culturable pseudomonads were enumerated from roots and soil of sorghum (Redlan and RTx433) and wheat (Lewjain) seedlings repeatedly grown in cycled soils in the growth chamber. Pseudomonads and Fusarium spp. were assessed from roots and soil of field-grown sorghum along with biological control traits hydrogen cyanide (HCN) and 2,4-diacetylphlorogluconol (phl) production. After four 4-week cycles, soil associated with Redlan seedlings had greater numbers of fluorescent pseudomonads than Lewjain. In dryland field conditions, RTx433 roots had greater numbers of pseudomonads than Redlan before anthesis but similar numbers after. There were no differences in numbers of pseudomonads from dryland soil or roots or soil of irrigated plants. Percentages of HCN-producing root isolates and phl soil isolates declined on irrigated Redlan plants, but percentages of HCN-producers increased in dryland conditions. Redlan roots had greater percentages of Fusarium isolates in the Gibberella fujikuroi complex. Results indicated that sorghum genotype affected root-associated populations of fluorescent Pseudomonas spp. and Fusarium spp. across soil environments.     

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.     

Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD⁺) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD⁺ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C_Ts) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD⁺ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD⁺ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r² = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

Plant genotype, micronutrient fertilization and take-all infection influence bacterial populations in the rhizosphere of wheat

The relationship between micronutrient efficiency of four wheat (Triticum aestivum L.) genotypes, tolerance to take-all disease (caused by Gaeumannomyces graminis (Sacc.) Arx and Olivier var. tritici Walker), and bacterial populations in the rhizosphere was tested in soil fertilized differentially with Zn and Mn. Plant growth was reduced by Mn or Zn deficiency and also by take-all. There was an inverse relationship between micronutrient efficiency of wheat genotypes when grown in deficient soils and the length of take-all lesions on roots (efficient genotypes had shorter lesions than inefficient ones). In comparison to the rhizosphere of control plants of genotypes Aroona and C8MM receiving sufficient Mn and Zn, the total numbers of bacterial cfu (colony forming units) were greater in the rhizosphere of Zn-efficient genotype Aroona under Zn deficiency and in Mn-efficient genotype C8MM under Mn deficiency. These effects were not observed in other genotypes. Take-all decreased the number of bacterial cfu in the rhizosphere of fully-fertilized plants but not of those subjected to either Mn or Zn deficiency. In contrast, the Zn deficiency treatment acted synergistically with take-all to increase the number of fluorescent pseudomonads in the rhizosphere. Although numbers of Mn-oxidising and Mn-reducing bacteria were generally low, take-all disease increased the number of Mn reducers in the rhizosphere of Mn-efficient genotypes Aroona and C8MM. Under Mn-deficiency conditions, the number of Mn reducers in the rhizosphere increased in Aroona but not in C8MM wheat. The results suggest that bacterial microflora may play a role in the expression of Mn and Zn efficiency and tolerance to take-all in some wheat genotypes.     

Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin

Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl⁺ Plt⁺ strains isolated from tobacco, cotton or wheat on different continents. Phl⁺ Plt⁺ strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the ‘P. syringae’, ‘P. fluorescens’ and ‘P. chlororaphis’ species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl⁺ Plt⁺ strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl⁺ strains, the Phl⁺ Plt⁺ strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl⁺ Plt⁺ fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0^T (=CFBP 6595^T, =DSM 19095^T) as the type strain.     

Incidence of inhibitory pseudomonads in the Pacific Northwest

Incidence of pseudomonads inhibitory to the root growth of till and no-till seeded crops winter wheat (Triticum aestivum L.), pea (Pisum sativum), lentil (Lens culinaris), and no-till winter barley (Hordeum vulgare), top and bottom of a seeded slope, and on the weed downy brome (Bromus tectorum) was investigated. Pseudomonads on the rhizoplane of these plants ranged from 10⁶ to 10⁸ colony-forming units (cfu) per gram dry weight of root. Neither tillage management nor site on a seeded slope affected colonizing numbers. Total numbers of pseudomonads were reduced in a second sampling, particularly on winter barley roots. However, more inhibitory pseudomonads were found in the second sampling. Several of the isolates, both inhibitory and stimulatory from different host plants, were bioassayed against winter wheat seedlings. Generally, the effect was different on the winter wheat than on the host plant indicating the organisms had some specificity. Several pseudomonads were isolated that severely reduced downy brome root growth and not that of winter wheat. Contribution from Agric. Res. Serv., U S Dep. of Agric., in cooperation with the College of Agric, and Home Economics, Agric. Res. Center, Washington State Univ., Pullman, WA 99164. Scientific Paper No. 7491.     

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.     

Bacterial associations with the mycorrhizosphere and hyphosphere of the arbuscular mycorrhizal fungus Glomus mosseae

Roots and mycorrhizal fungi may not associate with soil bacteria randomly, but rather in a hierarchical structure of mutual preferences. Elucidation of such structures would facilitate the management of the soil biota to enhance the stability of the plant-soil system. We conducted an experiment utilizing two isolates of soil bacteria to determine their persistence in distinct mycorrhizal regions of the root zone, and their effects on general rhizosphere populations of fluorescent pseudomonads (FP). Split-root sorghum (Sorghum bicolor L.) plants were grown in four-compartment containers, constructed so that the soils in individual compartments held either (1) roots colonized by the arbuscular-mycorrhizal (AM) fungus Glomus mosseae (M), (2) nonAM roots only (R), (3) hyphae of G. mosseae (H), or (4) no mycorrhizal structures (S). The soils were inoculated (10⁷ cells g^-1 dry soil) with antibiotic-resistant (rifampicin, rif; streptomycin, sm) strains of the soil bacteria, Alcaligenes eutrophus (rif^r50) or Arthrobacter globiformis (sm^r250), or were left uninoculated as control. A. eutrophus had been isolated from a specific source (hyphosphere soil of G. mosseae), and A. globiformis from mycorrhizosphere soils of two AM fungi. After 10 wk of growth, the presence of A. eutrophus was barely detectable (<10 cfu g^-1 dry soil) in nonAM (R and S) soils, but persisted well (10⁴ cfu g^-1 dry soil) in AM (H and M) soils. Numbers of A. globiformis were more evenly distributed between all soils, but were highest in the presence of AM roots (M soil). There were varied bacterial effects on root and AM-hyphal development: A. eutrophus decreased hyphal length in H soil, while A. globiformis stimulated root length in M soil. The two bacterial inoculants did not affect numbers of FP in H, R, and M soils, but the AM status of the soils did: the numbers of FP increased in the order M>R>H>S. There was a positive correlation of FP numbers with both bacterial inoculants in M and H soils. Numbers of FP changed with root or hyphal lengths, an effect that was related to changes in the numbers of the inoculated bacteria. The results indicate that the hyphosphere-specific A. eutrophus depended on the presence of G. mosseae, but that the nonspecific A. globiformis did not. The mycorrhizal status of soils may selectively influence persistence of bacterial inoculants as well as affecting the numbers of other native bacteria.     

Effects of Planting Date,Small Grain Crop Destruction,Fallow, and Soil Temperature on the Management of Meloidogyne incognita

The effects of planting date, rye (Secale cereale cv. Wren Abruzzi) and wheat (Triticura aestivum cv. Coker 797), crop destruction, fallow, and soil temperature on managing Meloidogyne incognita race 1 were determined in a 2-year study. More M. incognita juveniles (J2) and egg-producing adults were found in roots of rye planted 1 October than in roots of rye planted 1 November and wheat planted 1 November and 1 December. Numbers of M. incognita adults with and without egg masses were near or below detectable levels in roots of rye planted 1 November and wheat planted 1 November and 1 December. Meloidogyne incognita survived the mild winters in southern Georgia as J2 and eggs. The destruction of rye and wheat as a trap crop 1 March suppressed numbers of J2 in the soil temporarily but did not provide long-term benefits for susceptible crops that followed. In warmer areas where rye and wheat are grown in winter, reproduction of M. incognita may be avoided by delaying planting dates until soil temperature declines below the nematode penetration threshold (18 C), but no long-term benefits should be expected. The temperature threshold may be an important consideration in managing M. incognita population densities in areas having lower winter soil temperatures than southern Georgia.     

Role of ptsP, orfT, and sss Recombinase Genes in Root Colonization by Pseudomonas fluorescens Q8r1-96

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.     

Interplay between Wheat Cultivars,Biocontrol Pseudomonads,and Soil

There is a significant potential to improve the plant-beneficial effects of root-colonizing pseudomonads by breeding wheat genotypes with a greater capacity to sustain interactions with these bacteria. However, the interaction between pseudomonads and crop plants at the cultivar level, as well as the conditions which favor the accumulation of beneficial microorganisms in the wheat rhizosphere, is largely unknown. Therefore, we characterized the three Swiss winter wheat (Triticum aestivum) cultivars Arina, Zinal, and Cimetta for their ability to accumulate naturally occurring plant-beneficial pseudomonads in the rhizosphere. Cultivar performance was measured also by the ability to select for specific genotypes of 2,4-diacetylphloroglucinol (DAPG) producers in two different soils. Cultivar-specific differences were found; however, these were strongly influenced by the soil type. Denaturing gradient gel electrophoresis (DGGE) analysis of fragments of the DAPG biosynthetic gene phlD amplified from natural Pseudomonas rhizosphere populations revealed that phlD diversity substantially varied between the two soils and that there was a cultivar-specific accumulation of certain phlD genotypes in one soil but not in the other. Furthermore, the three cultivars were tested for their ability to benefit from Pseudomonas inoculants. Interestingly, Arina, which was best protected against Pythium ultimum infection by inoculation with Pseudomonas fluorescens biocontrol strain CHA0, was the cultivar which profited the least from the bacterial inoculant in terms of plant growth promotion in the absence of the pathogen. Knowledge gained of the interactions between wheat cultivars, beneficial pseudomonads, and soil types allows us to optimize cultivar-soil combinations for the promotion of growth through beneficial pseudomonads. Additionally, this information can be implemented by breeders into a new and unique breeding strategy for low-input and organic conditions.Improvement of plant fitness and yield by root-colonizing microorganisms is of special value in low-input or organic wheat production. Beneficial soil bacteria, such as certain Pseudomonas strains, are known to promote plant growth, which might help to circumvent potential negative consequences of low-input cropping systems, such as the limited supply of nutrients and higher disease pressure. A wide range of traits in Pseudomonas spp. are responsible for plant-beneficial effects. Many pseudomonads are capable of solubilizing poorly soluble or insoluble mineral phosphates, thereby rendering this element available for the plant and promoting plant growth (25, 43). Root-colonizing pseudomonads are also able to indirectly promote plant growth by providing protection against plant diseases. The most important mechanisms for plant protection against attacking pathogens are the induction of systemic resistance in plants (3) and the direct suppression of soilborne pathogens through the production of antimicrobial metabolites (16). The protection of wheat plants against Gaeumannomyces graminis var. tritici by naturally occurring pseudomonads in take-all decline soils is a well-described phenomenon and highlights the importance of these bacteria in a successful and environmentally friendly wheat production (53). Interestingly, in many naturally disease-suppressive soils a specific group of fluorescent pseudomonads is enriched, which is able to produce the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) (6, 38, 53). The production of the polyketide DAPG, which has broad-spectrum activity against bacteria, plants, fungi, and nematodes (8, 9, 21, 28, 33, 45), has been shown to be a key factor in the suppression of soilborne plant diseases by various Pseudomonas biocontrol strains (16).The degree of plant protection and plant growth promotion provided by root-colonizing pseudomonads is highly dependent on different environmental factors. For example, the expression of important biocontrol genes such as DAPG or HCN biosynthetic genes in the rhizosphere is modulated by biotic factors such as fungi and other bacteria present in the rhizosphere and the secondary metabolites they release (7, 19, 27, 29, 32). Moreover, it has been observed that the plant species and cultivar as well as the physiological stage of the plant can influence the expression of biocontrol genes and the production of antimicrobial metabolites (4, 7, 19, 32, 35). In addition to the production of DAPG and other antimicrobial metabolites, efficient colonization of roots is a prerequisite for beneficial plant-Pseudomonas interactions. Root colonization is dependent not only upon specific characteristics of the bacterium itself but also on root morphology and root exudates that vary between host plant species and even between cultivars of the same species (5, 34). The host species/cultivar also influences the abundance and diversity of naturally occurring pseudomonads (13). This has been shown in particular for DAPG-producing populations (4, 5, 26, 30, 36).Wheat is a crop known to benefit strongly from naturally occurring DAPG-producing pseudomonad populations (52). It has been shown that the size and composition of DAPG-producing populations in the wheat rhizosphere and also the amount of DAPG produced by these populations may vary substantially between different cultivars (4, 35). However, holistic studies which evaluate specific wheat cultivars for both their ability to benefit from plant growth-promoting pseudomonads and their influence on bacterial populations and production of biocontrol compounds are missing. A comprehensive characterization of different cultivars is needed in order to better understand which cultivars promote beneficial interactions with the pseudomonads. This knowledge has potential in future breeding strategies to be used for selection of new cultivars that optimally attract and respond to these bacteria.In order to address this gap in knowledge, this study evaluated three Swiss winter wheat (Triticum aestivum) cultivars for several characteristics considered important in a successful wheat-pseudomonas interplay: (i) the ability to accumulate pseudomonads and phlD⁺ pseudomonads in two different Swiss soils, (ii) the ability to select for individual phlD⁺ genotypes in two different soils, (iii) the ability to benefit from the two model biocontrol strains, Pseudomonas fluorescens strain CHA0 (a DAPG producer) and P. putida KD (a DAPG nonproducer), in terms of direct plant growth promotion and disease suppression, and finally (iv) the level of biocontrol gene expression (DAPG-biosynthetic gene phlA) in the rhizosphere.     

Soil conduciveness to take-all of wheat: Influence of the nitrogen fertilizers on the structure of populations of fluorescent pseudomonads

In a field cropped with wheat, a high and low level of soil conduciveness to take-all were induced by applying a nitrogen fertilizer with either calcium nitrate or ammonium sulphate. From these two soils, two representative populations of fluorescent pseudomonads were tested for their in situ behaviour. Take-all index and root dry weight were assessed on plants cropped in soils infested with Gaeumannomyces graminis var tritici (Ggt) and each bacterized with one of the isolates of fluorescent pseudomonads. The bacteria tested can be split into three groups: antagonists which reduce take-all, deleterious isolates which aggravate the disease and neutral without evident effect on the disease. The predominance of antagonistic fluorescent pseudomonads in the NH₄-treated soil and the predominance of deleterious ones in the NO₃-treated soil was confirmed after statistical analysis. The microbial impact on take-all must be more considered as the resulting effect of divergent activities of both rhizobacteria types than the only consequences of the presence of antagonistic pseudomonads. All the high cyanogenic pseudomonads were antagonists in situ and were more numerous in the NH₄-treated soil than in the NO₃-treated soil.     

Cross Talk between 2,4-Diacetylphloroglucinol-Producing Biocontrol Pseudomonads on Wheat Roots

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a ΔphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.     

Identification and Specific Detection of a Novel Pseudomonadaceae Cluster Associated with Soils from Winter Wheat Plots of a Long-Term Agricultural Field Experiment

The genus Pseudomonas (sensu stricto) represents a group of microorganisms directly involved in functions conferring plant health. We performed a study in the DOK long-term agricultural field experiment on the basis of previously published Pseudomonas-selective PCR primers in order to investigate the community structure of the microbial groups defined by the target range of these primers. Three different agricultural management systems, i.e., conventional, biodynamic, and bio-organic, along with mineral and unfertilized controls were investigated, with each system planted with either winter wheat or a grass-clover ley. Amplified small-subunit rRNA gene fragments were analyzed using the genetic profiling techniques restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE), revealing distinct differences between soils planted with winter wheat and grass clover but only minor differences between the management systems. Phylogenetic analyses of 59 clone sequences retrieved from bio-organic and unfertilized systems identified sequences related to Pseudomonas fluorescens and a novel cluster termed Cellvibrio-related Pseudomonadaceae (CRP). The CRP clones were exclusively isolated from winter wheat soil samples and were responsible for the crop-specific differences observed in RFLP and DGGE profiles. New primers were designed for the amplification of CRP targets directly from soil DNA, yielding strong signals exclusively for winter wheat soils. We concluded that crop-associated CRP exist in agricultural soils and that genetic profiling followed by specific probe design represents a valuable approach for identification as well as sensitive and rapid monitoring of novel microbial groups in the environment.     

Biological Control of Meloidogyne hapla on Alfalfa and Tomato with the Fungus Meria coniospora

This study was to determine whether Arthrobotrys flagrans, A. oligospora, and Meria coniospora would control the root-knot nematode Meloidogyne hapla on alfalfa and tomato. Alfalfa seeds were coated with a fungus-rye powder in 2% cellulose and were planted in infested soil. Three-week-old seedlings from seed treated with M. coniospora had 60% and 58% fewer galls in two experiments than did seedlings from untreated seeds. Numbers of J2 in the soil were not reduced. Plant growth did not improve. When seed of tomato were coated with M. coniospora and planted in M. hapla-infested soil, roots had 34% fewer galls and 47% fewer J2 in the soil at 28 days. After 56 days there was no reduction in J2 numbers. Plant growth did not improve. When roots of tomato transplants were dusted with M. coniospora fungus-rye powder or sprayed with a spore suspension before planting in M. hapla-infested soil, 42% and 35%, respectively, fewer galls developed in 28 days on treated roots than on roots not treated with fungus. The numbers of J2 extracted from roots or recovered from soil were not reduced, however, and plant growth did not improve.     

Efficacy of Bacillus thuringiensis,Paecilomyces marquandii,and Streptomyces costaricanus with and without Organic Amendments against Meloidogyne hapla Infecting Lettuce

Chitin, wheat mash, or brewery compost were incorporated into unfumigated and methyl bromide-fumigated organic soils placed in microplots formed from cylindrical drainage tiles (0.25 m-diam. clay tile). After 3 weeks, Meloidogyne hapla and cell or spore suspensions of Bacillus thuringiensis, Paecilomyces marquandii, and Streptomyces costaricanus were individually added to the soils of designated microplots. A B. thuringiensis + S. costaricanus combination was also tested. Lettuce seedlings, cv. Montello, were transplanted into the soils 3 to 4 days later. All the bacterial and fungal antagonists applied without a soil amendment, except the B. thuringiensis + S. costaricanus treatment, reduced root galling and increased lettuce head weight in the unfumigated organic soil, but not in the fumigated soil. All three amendments were also effective against M. hapla and reduced root galling in fumigated and unfumigated soils. Wheat mash amendment increased lettuce head weight in the unfumigated soil. In general, no antagonist × amendment interaction was detected. Soil populations of B. thuringiensis were maintained at ≥4.0 log10 colony-forming units/g organic soil during the first 14 days after planting. However, viable cells of B. thuringiensis were not detected after 49 days.     

Influence of agricultural practices and seasons on the abundance and community structure of culturable pseudomonads in soils under no-till management in Argentina

Background and aims

Members of the genus Pseudomonas are common inhabitants of rhizospheres and soils, and it is known that soil types and crop species influence their population density and structure. 20?×?10⁶ ha are cultivated under no-tillage in Argentina and there is a need to find new biologically-based soil quality indexes to distinguish between sustainable and non-sustainable agricultural practices. Pseudomonads abundance and community structure were analyzed in no-till soils with different agricultural practices, in productive fields along 400 km of Argentinean Pampas.

Methods

We sampled soils and root systems from agricultural plots in which sustainable or non-sustainable agricultural practices have been applied. Samples were collected in summer and winter during 2010 and 2011. Culturable fluorescent and total pseudomonads were enumerated by plating on Gould’s selective medium S1. Colonies from these plates served as DNA source to carry out PCR-RFLP community structure analysis of the pseudomonads-specific marker genes oprF and gacA.

Results

Abundance of total and fluorescent culturable pseudomonads in bulk soils was influenced by seasonal changes and agricultural practices. Rhizospheric counts from the same crop were affected by agricultural treatments. Also, crop species influenced pseudomonads density in the rhizosphere. Combined PCR-RFLP profile of both genes showed a seasonal grouping of samples.

Conclusions

Sustainable soil management seems to promote pseudomonads development in soils, favoring root colonization of crops from those plots. Crop species influence total pseudomonads load of rhizospheres and its community structure. Total or relative pseudomonads load could function as soil quality indicator of good agricultural practices.     

Growth characteristics of ectomycorrhizal seedlings of Quercus glauca,Quercus salicina,and Castanopsis cuspidata planted on acidic soil

Key message

Seedlings of three Fagaceae species planted on acidic, infertile colluvial soil showed accelerated growth when inoculated with ectomycorrhizal fungi.

Abstract

We conducted a study with seedlings of Fagaceae species inoculated with ectomycorrhizal fungi to estimate their utility for growth in acidic soil conditions. We selected Quercus glauca, Quercus salicina and Castanopsis cuspidata as typical evergreen, broad-leaved, woody species of southwestern Japan. Seedlings were inoculated with Astraeus hygrometricus or Scleroderma citrinum, and planted in acidic, infertile colluvial soil collected from an abandoned site. Six months after planting, seedlings of the three species inoculated with A. hygrometricus were growing well, especially, Q. salicina. The growth of seedlings inoculated with S. citrinum was inferior to seedlings inoculated with A. hygrometricus. In contrast, seedlings without ectomycorrhizal fungi did not grow well. Differences in growth among the three types of seedlings were related to differences in the levels of nutrient acquisition. We concluded that Fagaceae seedlings inoculated with A. hygrometricus were best suited for acidic, infertile environments.     

Relationships between take-all,soil conduciveness to the disease,populations of fluorescent pseudomonads and nitrogen fertilizers

Take-all of wheat, caused by Gaeumannomyces graminis var tritici (Ggt), is reduced by ammoniacal fertilizers as compared to nitrate sources. This influence of nitrogen on the disease is only observed on nodal roots at flowering. But soil conduciveness to take-all, as measured in a soil bioassay, is modified earlier. Forty days after nitrogen application at early tillering, the NH₄-treated soil became less conducive than the NO₃-treated one. When nitrogen applications are done at sowing and at tillering, differences in disease propagation between the two soils are enhanced. Results from four years of experimentation show that when the level of natural soil inoculum is high, disease severity is reduced by ammonium, showing an effect on the parasitic phase of Ggt. At a low level of natural inoculum the effect of the source of nitrogen is mainly observed on the percent of infected plants, indicating that the saprophytic and preparasitic phases are affected. Rhizospheric bacterial populations increase from sowing to tillering, but differences on take-all conduciveness after tillering are not correlated with differences in the amounts of aerobic bacteria or fluorescent pseudomonads isolated from soils treated with different sources of nitrogen. Qualitative changes in fluorescent Pseudomonas spp. populations, like in vitro antagonism, are more likely to explain differences in soil conduciveness to take-all than are quantitative changes in this group. Nevertheless, the introduction of Ggt in a cropped soil leads to a greater increase in fluorescent pseudomonads populations than in total aerobic bacteria.The delay between reducing soil conduciveness and reducing disease in the field with ammonium nitrogen fertilization, the qualitative change of fluorescent pseudomonads populations and the role of necroses in rhizobacteria multiplication, provide information leading to our representation of a dynamic model based on the differentiation of the wheat root system into seminal and nodal roots.     

Ectomycorrhizal inoculum potential of northeastern US forest soils for American chestnut restoration: results from field and laboratory bioassays

American chestnut (Castanea dentata) was once a dominant overstory tree in eastern USA but was decimated by chestnut blight (Cryphonectria parasitica). Blight-resistant chestnut is being developed as part of a concerted restoration effort to bring this heritage tree back. Here, we evaluate the potential of field soils in the northern portion of the chestnut's former range to provide ectomycorrhizal (EM) fungus inoculum for American chestnut. In our first study, chestnut seedlings were grown in a growth chamber using soil collected from three sites dominated by red oak (Quercus rubra) as inoculum and harvested after 5 months. Of the 14 EM fungi recovered on these seedlings, four species dominated in soils from all three sites: Laccaria laccata, a Tuber sp., Cenococcum geophilum, and a thelephoroid type. Seedlings grown in the nonsterilized soils were smaller than those growing in sterilized soils. In the second study, chestnut seedlings were grown from seed planted directly into soils at the same three sites. Seedlings with intermingling roots of established trees of various species were harvested after 5 months. Seventy-one EM fungi were found on the root tips of the hosts, with 38 occurring on chestnut seedlings. Multiple versus single host EM fungi were significantly more abundant and frequently encountered. The fungi observed dominating on seedlings in the laboratory bioassay were not frequently encountered in the field bioassay, suggesting that they may not have been active in mycelial networks in the field setting but were in the soils as resistant propagules that became active in the bioassay. These results show that soil from red oak stands can be used to inoculate American chestnut with locally adapted ectomycorrhizal fungi prior to outplanting, a relatively cost effective approach for restoration efforts.