Diversity and antagonistic potential of Pseudomonas spp. associated to the rhizosphere of maize grown in a subtropical organic farm

Pseudomonas spp. are one of the most important bacteria inhabiting the rhizosphere of diverse crop plants and have been frequently reported as biological control agents (BCAs). In this work, the diversity and antagonistic potential of Pseudomonas spp. in the rhizosphere of maize cultivars Nitroflint and Nitrodent grown at an organic farm in Brazil was studied by means of culture-dependent and -independent methods, respectively. Sampling of rhizosphere soil took place at three different stages of plant development: 20, 40 and 106 days after sowing. A PCR-DGGE strategy was used to generate specific Pseudomonas spp. fingerprints of 16S rRNA genes amplified from total community rhizosphere DNA. Shifts in the relative abundance of dominant populations (i.e. PCR-DGGE ribotypes) along plant development were detected. A few PCR-DGGE ribotypes were shown to display cultivar-dependent relative abundance. No significant differences in diversity measures of DGGE fingerprints were observed for different maize cultivars and sampling times. The characterisation and assessment of the antagonistic potential of a group of 142 fluorescent Pseudomonas isolated from the rhizosphere of both maize cultivars were carried out. Isolates were phenotypically and genotypically characterised and screened for in vitro antagonism towards three phytopathogenic fungi and the phytopathogenic bacterium Ralstonia solanacearum. Anti-fungal activity was displayed by 13 fluorescent isolates while 40 isolates were antagonistic towards R. solanacearum. High genotypic and phenotypic diversity was estimated for antagonistic fluorescent Pseudomonas spp. PCR-DGGE ribotypes displayed by antagonists matched dominant ribotypes of Pseudomonas DGGE fingerprints, suggesting that antagonists may belong to major Pseudomonas populations in the maize rhizosphere. Antagonists differing in their genotypic and phenotypic characteristics shared the same DGGE electrophoretic mobility, indicating that an enormous genotypic and functional diversity might be hidden behind one single DGGE band. Cloning and sequencing was performed for a DGGE double-band which had no corresponding PCR-DGGE ribotypes among the antagonists. Sequences derived from this band were affiliated to Pseudomonas stutzeri and P. alcaligenes 16S rRNA gene sequences. As used in this study, the combination of culture-dependent and -independent methods has proven to be a powerful tool to relate functional and structural diversity of Pseudomonas spp. in the rhizosphere.     

Fungal diversity in agricultural soil under different farming management systems,with special reference to biocontrol strains of Trichoderma spp.

Fungal communities and their dynamics were investigated in relation to season, soil type and farming management practice. The research was done using soils from high- (H) and low-yield areas (L) of a field site cultivated with winter wheat under two different farming management systems (precision farming, P; conventional farming, C) over the vegetation period. Fungal diversity was analysed by cultivation-independent methods [direct extraction of DNA from soil followed by PCR amplification of a subunit of the 18S rDNA and fingerprinting (DGGE)] as well as cultivation-dependent techniques (isolation of pure cultures). The comparisons of the PCR amplicons by DGGE patterns showed no differences between the different sampling sites and no influence of the farming management systems. Only small differences were observed over the vegetation period. For cultivation purposes active hyphae were isolated using a soil-washing technique. The resulting isolates were subcultured and grouped by their morphology and genotype. In contrast to the cultivation independent approaches, clear site-specific and seasonal effects on the fungal community structure could be observed. However, minor effects of the different farming management techniques applied were visible for active populations. These results clearly indicate that the potential fungal community (including spores), investigated by the cultivation-independent approach, is almost entirely uninfluenced by the investigated factors, whereas active populations show a clear response to environmental changes. The most abundant group consisting of Trichoderma species was investigated in more detail using strain-specific genotype based fingerprinting techniques as well as a screening for potential biocontrol activity against the wheat pathogen Fusarium graminearum. The genotypic distribution as well as the potential biocontrol activity revealed clear site-specific patterns.     

Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop

We assessed the Azospirillum inoculation and N-fertilization effect on grain yield and on the phyllosphere endophytic diversity of nitrogen-fixing bacteria in a rice rainfed crop. We used cultivation-based techniques and cultivation-independent methods involving PCR-16S rRNA and denaturing gradient gel electrophoresis (DGGE). In general, we observed that grain yield was improved when inoculated with Azospirillum (depending on the genotype) and/or fertilized with urea. A similar behavior was observed in total N-content in grain and the MPN determination, as the highest values occurred when seeds were inoculated with A. brasilense REC3 (S1) than with A brasilense 13-2C (S2). A positive nitrogenase activity and PCR-nifH amplification suggests that the bacteria associated to inner tissues of rice phyllosphere could have contributed to the different N-contents detected. The bacterial diversity, observed in the number and intensity of DGGE profiles, showed a higher number of bands when total DNA was obtained using only CTAB than with CTAB + PVP. The DGGE profiles revealed great stability in the dominating bands, which presumably represent numerically dominant species. Application of A. brasilense strains as inoculants did not influence the dominant members of the endophytic microbial communities in the phyllosphere, but improved N-content and production of rainfed rice crop.     

Association between Burkholderia species and arbuscular mycorrhizal fungus spores in soil

Burkholderia pseudomallei, the bacterial cause of the potentially fatal infection known as melioidosis, has a facultative intracellular lifestyle. The intracellular presence of B. pseudomallei in various eukaryotes including arbuscular mycorrhizal fungus (AMF) spores can be demonstrated in vitro. AMF spores were isolated from soils in a melioidosis-endemic area. B. pseudomallei and other Burkholderia spp. DNA was detected in these AMF spore samples, confirming an AMF spore-Burkholderia spp. association in soils which did not yield Burkholderia spp. by culture. This association may explain the environmental persistence, difficulty of recovery and dispersal of Burkholderia spp. in specific environments.     

Changes in agricultural management drive the diversity of Burkholderia species isolated from soil on PCAT medium

In order to assess the diversity of culturable Burkholderia populations in rhizosphere and bulk soil and to evaluate how different agricultural management regimes and land use history affect this diversity, four treatments were evaluated: permanent grassland; grassland converted into maize monoculture; arable land and arable land converted into grassland. Burkholderia isolates obtained on PCAT medium were grouped in 47 clusters using 16S ribosomal RNA gene based PCR-DGGE combined with BOX genomic fingerprinting (DGGE-BOX). The distribution of the isolates in the DGGE-BOX clusters was used to calculate the Shannon diversity index per treatment. Interestingly, we observed that the Burkholderia diversity was affected by changes in the agricultural management, since the highest diversity was observed in permanent grassland and in continuous arable land. In addition, the diversity tended to be higher in the rhizosphere than in the corresponding bulk soil. The use of species abundance models indicated that rhizosphere communities had more even distributions than communities collected from the bulk soil. Identification of isolates revealed that only 2% of these belonged to the B. cepacia complex and that the majority was assigned to either (1) new Burkholderia species or (2) Burkholderia species that had originally been isolated from soil. Isolates classified as B. hospita, B. caledonica and Burkholderia sp. ‘LMG 22934’ and ‘LMG 22936’ were found mainly in the arable land, while isolates belonging to Burkholderia sp. ‘LMG 22929’ and B. phytofirmans were associated with the grassland area. Another potentially new Burkholderia species, ‘LMG 22932’, was found in both areas, in close association with the maize rhizosphere.     

Zinc uptake by mycorrhizal and uninfected roots of Pinus radiata and Araucaria cunninghamii

Uptake of zinc from solution by uninfected roots, ectomycorrhizas of Pinus radiata, and endomycorrhizas of Araucaria cunninyhamii occurred by a non-metabolic phase and by metabolically mediated absorption. The metabolically mediated absorption of Zn was 1.4–4.5 times greater for ectomycorrhizas than for uninfected roots and 2.6 times greater for endomycorrhizas than for uninfected roots. Part of the increase with ectomycorrhizas was due to their larger size than uninfected roots and when corrected for this, the uptake/cm² was 1.4–3.0 times greater. The roles of mycorrhizas in uptake of Zn and other trace elements from soil are discussed.     

Hydrocarbon degradation potential and activity of endophytic bacteria associated with prairie plants

Phytoremediation systems for organic compounds such as petroleum hydrocarbons rely on a synergistic relationship between plants and their root-associated microbial communities. To determine the probable role of endophytic bacterial communities in these systems, this study examined both rhizosphere and endophytic communities of five different plant species at a long-term phytoremediation field site. Hydrocarbon degradation potential and activity were assessed using MPN assays, PCR analysis of catabolic genes associated with hydrocarbon degradation, and mineralization assays with C-14 labeled hydrocarbons. Microbial community structure in each niche was assessed by DGGE analysis of 16S rRNA gene fragments and subsequent band sequencing. Both endophytic degrader populations and endophytic degrader activity showed substantial inter-species variation, largely independent of that shown by the respective rhizosphere populations. Endophytic hydrocarbon degradation was linked to dominant bacterial endophytes. Pseudomonas spp. dominated endophytic communities exhibited increased alkane hydrocarbon degradation potential and activity, while Brevundimonas and Pseudomonas rhodesiae dominated endophytic communities were associated with increased PAH degradation potential and activity. In one plant species, Lolium perenne, increased endophytic alkane hydrocarbon degradation was associated with increased rhizosphere alkane degradation and decreased rhizosphere PAH degradation. Our results show that diverse plant species growing in weathered-hydrocarbon contaminated soil maintain distinct, heterogeneously distributed endophytic microbial populations, which may impact upon the ability of plants to promote the degradation of specific types of hydrocarbons.     

Seasons differently impact the structure of mineral weathering bacterial communities in beech and spruce stands

Bacterial communities play an essential role in the sustainability of forest ecosystems by releasing from soil minerals the nutritive cations required not only for their own nutrition but also for that of trees. If it is admitted that the nutritional needs of trees vary during seasons, the seasonal dynamics of the mineral weathering bacterial communities colonizing the tree rhizosphere remain unknown. In this study, we characterized the mineral weathering efficacy of bacterial strains, from the rhizosphere and the adjacent bulk soil at four different seasons under two different tree species, the evergreen spruce and the deciduous beech, using a microplate assay that measures the quantity of iron released from biotite. We showed that the functional and taxonomic structures of the mineral weathering bacterial communities varied significantly with the tree species as well as with the season. Notably, the Burkholderia strains from the beech stand appeared more efficient to weather biotite that the one from the spruce stand. The mineral weathering efficacy of the bulk soil isolates did not vary during seasons under the beech stand whereas it was significantly higher for the spring and summer isolates from the spruce stand. The weathering efficacy of the rhizosphere isolates was significantly higher for the autumn isolates compared to the isolates sampled in the other seasons under the beech stand and in summer compared to the other seasons under spruce. These results suggest that seasonal differences do occur in forest soil bacterial communities and that evergreen and deciduous trees do not follow the same dynamic.     

Responses of oribatid mites to tree girdling and nutrient addition in boreal coniferous forests

Tree girdling has been experimentally used to stop the allocation of carbohydrates from the canopy to tree roots and their associated ectomycorrhizal (EM) mycelia. We used three already established girdling experiments in northern Sweden, one in a Pinus sylvestris stand and two in Picea abies stands, to determine the effect of tree girdling on soil-living oribatid mites. These mites often feed on fungal hyphae, but it is not known to what extent they feed on EM or saprotrophic fungi. We hypothesised that a presumed decline in EM fungi after girdling would strongly reduce EM-feeding specialists and, correspondingly, reduce total abundance and species richness of oribatids in girdled plots. Tree girdling resulted in a significant decline in total abundance of oribatid mites in the two spruce stands, which was assumed to be linked to the decline in EM fungi, but not in the pine stand. Species richness decreased in girdled plots in one of the spruce forests. The decline in total abundance in girdled spruce stands was primarily dependent on a significant and consistent population decrease in Oppiella nova, which was the most abundant oribatid mite in the ungirdled spruce plots. Its abundance after girdling was only 8–18% of that in ungirdled spruce plots. In the pine stand, O. nova had much lower abundance than in the spruce stands, and despite a tendency to decline in number after girdling also in the pine stand, it had a minor effect on total abundance. These results suggest that O. nova may be dependent on EM fungi in spruce forest soils, whereas the dependence on EM fungi in pine forest soils is less evident.     

Impact of incorporated fresh 13C potato tissues on the bacterial and fungal community composition of soil

Soil microbial communities play a major role in organic matter decomposition, however the importance of the individual species involved is still unclear. To identify the dynamics and identity of bacterial species involved in decomposition of potato tissue as well as the assimilation of carbon from fresh plant material, ¹³C-labeled green potato tissues (¹³C 99.2%) were incorporated in soil microcosm for 39 days at the level of 2.5% (w/dry weight soil). The DNA was extracted from the soil after 1, 6, 15, 25 and 39 days. The heavy (¹³C) and light (¹²C) fractions of DNA were separated by ultracentrifugation and the structures of the bacterial and fungal communities were characterized by DGGE. Primary and secondary ¹³C-sequestrators were identified by sequencing DGGE bands that had appeared only in the heavy DNA fractions. Over the course of the experiment, the most dominant ¹³C-labeled phylogenetic group (class or phylum) was γ-Proteobacteria (51.4%), followed by Actinobacteria (27%), β-Proteobacteria (8.1%) and α-Proteobacteria (5.4%). Two taxa, namely Firmicutes and Verrucomicrobia, were represented by just one sequence type. These bacterial taxa were differentiated into primary (Arthrobacter, Pseudomonas) and secondary sequestrators (Actinobacteria, Dyella, Mesorhizobium and Sphingomonas). The latter were possibly involved in either the redistribution of previously consumed carbon or in a possible degradation of the more complex plant compounds. On the basis of this analysis, only 5 to 8 bacterial taxa were involved in carbon sequestration at any one measured time point. Our results show the importance of specific microbial taxa in the decomposition and mineralization of plant residues in soil, which will allow us to better understand the role of such communities in carbon cycling.     

Ectomycorrhizal Fungi and Associated Bacteria Provide Protection Against Heavy Metals in Inoculated Pine (Pinus Sylvestris L.) Seedlings

The roles of ectomycorrhizal fungi and bacteria associated with corresponding fungal species in distribution of heavy metals within roots and shoots of inoculated pine (Pinus sylvestris L.) seedlings were determined in this study. The mycorrhizal fungi forming different morphotypes were identified by PCR-RFLP using respective primers for an internal spacer transcribed region (ITS) of fungal rDNA. Amongst five fungal species detected, three were identified as Scleroderma citrinum, Amanita muscaria and Lactarius rufus. These fungi used for inoculation of pine seedlings significantly reduced translocation of Zn(II), Cd(II) or Pb(II) from roots to shoots, and the pattern of metal-accumulation was dependent on the fungal species. Ectomycorrhizae-associated bacteria identified as Pseudomonas were used as an additional component of the pine inoculation. These dual root inoculations resulted in higher accumulation of the metals, especially Zn(II), in the roots compared to the inoculation with fungal species alone. Consequently, dual inoculation of pine seedlings could be a suitable approach for plant protection against heavy metals and successful planting of metal-polluted soils.     

Interaction among free-living N-fixing bacteria isolated from Drosera villosa var. villosa and AM fungi (Glomus clarum) in rice (Oryza sativa)

Rice is usually grown in N-deficient soils, demanding that the element be supplied to the field by commercially available N fertilizers. Unfortunately, a substantial amount of the urea-N or NO₃-N applied as fertilizers is lost through different mechanisms, causing environmental pollution problems. Utilization of biological N₂ fixation (BNF) technology can decrease the application of N fertilizers, reducing environmental risks. This study evaluated the effects of four free-living N-fixing bacterial species, isolated from oligotrophic soil conditions, as single inoculants or combined with arbuscular mycorrhizal fungi (Glomus clarum), on the development of rice plants grown as flooded or upland rice, in the greenhouse. Upland rice roots were inoculated with Methylobacterium sp., Burkholderia sp. and Sphingomonas sp., whereas the species Burkholderia sp., Pseudomonas sp. and Sphingomonas sp., were inoculated on flooded rice. Inoculants consisted of individual bacterial species or their mixtures, with or without G. clarum. Controls included non-bacteria/non-AM fungi, and AM fungi alone. Experiments were carried out in five replicates. The presence of G. clarum decreased or did not significantly affect plant growth under the different culture conditions. The presence of AM fungi stimulated the N-fixing bacterial population of upland rice. Bacterial species had different effects, under both culture conditions, and some genera of N-fixing bacteria increased root and shoot growth at different plant growth stages. The level of mycorrhiza colonization had no influence on plant growth     

Timber harvesting alters soil carbon mineralization and microbial community structure in coniferous forests

Timber harvesting influences both above and belowground ecosystem nutrient dynamics. Impact of timber harvesting on soil organic matter (SOM) mineralization and microbial community structure was evaluated in two coniferous forest species, ponderosa pine (Pinus ponderosa) and lodgepole pine (Pinus contorta). Management of ponderosa pine forests, particularly even-aged stand practices, increased the loss of CO₂-C and hence reduced SOM storage potential. Changes in soil microbial community structure were more pronounced in ponderosa pine uneven-aged and heavy harvest stands and in lodgepole pine even-aged stand as compared to their respective unmanaged stands. Harvesting of trees had a negative impact on SOM mineralization and soil microbial community structure in both coniferous forests, potentially reducing coniferous forest C storage potential.     

Physiological, biochemical and molecular responses of the soil microbial community after afforestation of pastures with Pinus radiata

Afforestation and deforestation are key land-use changes across the world, and are considered to be dominant factors controlling ecosystem functioning and biodiversity. However, the responses of soil microbial communities to these land-use changes are not well understood. Because changes in soil microbial abundance and community structure have consequences for nutrient cycling, C-sequestration and long-term sustainability, we investigated impacts of land-use change, age of stand and soil physico-chemical properties on fungal and bacterial communities and their metabolic activities. This study was carried out at four sites in two geographical locations that were afforested on long-established pastures with Pinus radiata D. Don (pine). Two of the sites were on volcanic soils and two on non-volcanic soils and stand age ranged from 5 to 20 y. Microbial communities were analysed by biochemical (phospho-lipid fatty acids; PLFA) and molecular (multiplex-terminal restriction fragment length polymorphism; M-TRFLP) approaches. Both site and stand age influenced microbial properties, with changes being least detectable in the 5-y-old stand. Land use was a key factor influencing soil metabolic activities as measured by physiological profiling using MicroResp. Pasture soils had higher microbial biomass (P < 0.001), and metabolic activities (P < 0.001), and basal respiration rates were up to 2.8-times higher than in the pine soils. Microbial abundance analysis by PLFA showed that the fungal to bacterial ratio was higher in the pine soils (P < 0.01). Community analysis suggested that soil bacterial communities were more responsive to site (principal component 1; P < 0.001) than to land use (principal component 5; P < 0.001). In contrast, the fungal community was more affected by land-use change (principal component 1; P < 0.001) than by site, although site still had some influence on fungal community structure (principal component 2; P < 0.001). Redundancy analysis also suggested that bacterial and fungal communities responded differently to various soil abiotic properties, land-use change and location of sites. Overall, our results indicate that the change in land use from pasture to P. radiata stands had a direct impact on soil fungal communities but an indirect effect, through its effects on soil abiotic properties, on bacterial communities. Most of the changes in bacterial communities could be explained by altered soil physico-chemical properties associated with afforestation of pastures.     

Lichens and bryophyte communities of planted and semi-natural forests in Britain: the influence of site type, stand structure and deadwood

Lichen and bryophyte communities of spruce and pine plantations in different parts of Britain were surveyed and compared to those of semi-natural pine and oak woodlands. In total, 202 lichen species and 111 bryophytes were recorded. Community composition and species-richness were related to measures of climate, stand structure and deadwood (snags, logs and stumps). Plantations had a less species-rich lichen flora than semi-natural stands related to reduced light availability and lack of old trees. Bryophyte species-richness was similar in plantations and semi-natural stands, and was positively correlated with large diameter (>20 cm), well-decayed logs and stumps. Lichens species-richness was higher on decorticate snags (especially in semi-natural Scots pine stands in the Scottish Highlands). Early successional stands were often the richest for lichens, stumps being important for Calicium and Cladonia species. Three strategies are suggested for enhancing lower plant diversity in planted forests: (1) extending felling rotations; (2) introducing alternative silvicultural systems to clear-felling (e.g. single-tree selection) to foster continuity of woodland conditions and increase deadwood volumes; (3) modifying restocking practices on clear-fells to avoid excessive shading of deadwood.     

No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types

In boreal forests ericaceous shrubs often dominate the forest floor vegetation. Nitrogen enrichment has been shown to decrease shrub abundance and in this study we explored whether it also affects the root associated fungal communities. Fine roots of Vaccinium myrtillus were collected in a Norway spruce dominated forest and of Vaccinium vitis-idaea in a Scots pine dominated forest. In both forests, nitrogen enrichment was experimentally induced by adding 12.5 and 50 kg N ha⁻¹ yr⁻¹ for 12 (spruce forest) and four (pine forest) years. Based on terminal restriction fragment length polymorphisms, subcloning and sequencing analyses, the root associated fungal communities were examined. We found 93 fungal species including Asco-, Basidio- and Zygo-mycota. In general, the Rhizoscyphus ericae aggregate was the most dominant and this was followed by Herpotrichiellaceae and Sebacina. Ordination analysis revealed that nitrogen enrichment did not change species composition of the fungal communities in neither the spruce nor the pine forest, while fungal community structures were clearly discriminated between the dominant shrub species in each forest. Similarly, no fungal species showed a significant response to nitrogen enrichment. Therefore, nitrogen enrichment appears to have no effect on root associated fungi of understorey dwarf shrubs in boreal forests, while it is clear that spruce and pine forests harbor distinctive communities of these fungi.     

Evaluation of bacterial strains isolated from oil-contaminated soil for production of polyhydroxyalkanoic acids (PHA)

Samples of eight geographically distinct soils contaminated with crude oil were screened for polyhydroxyalkanoic acid (PHA) producing bacterial strains. Twenty three bacterial strains were able to accumulate PHA when sodium gluconate or sodium octanoate was used as the sole carbon source. Biochemical tests and 16S rRNA sequencing identified bacteria of the genera Pseudomonas, Acinetobacter, Sphingobacterium, Brochothrix, Caulobacter, Ralstonia, Burkholderia and Yokenella. Three of the bacterial strains have never been reported to produce PHA. The phylogenetic analysis of the PHA synthase (phaC) gene of these bacteria showed a close homology with the phaC gene of different Pseudomonas species.This study indicates that stressed environments like oil-contaminated sites can be potential sources of medium-chain-length PHA producers.     

Evaluation of bacterial strains isolated from oil-contaminated soil for production of polyhydroxyalkanoic acids (PHA)

Samples of eight geographically distinct soils contaminated with crude oil were screened for polyhydroxyalkanoic acid (PHA) producing bacterial strains. Twenty three bacterial strains were able to accumulate PHA when sodium gluconate or sodium octanoate was used as the sole carbon source. Biochemical tests and 16S rRNA sequencing identified bacteria of the genera Pseudomonas, Acinetobacter, Sphingobacterium, Brochothrix, Caulobacter, Ralstonia, Burkholderia and Yokenella. Three of the bacterial strains have never been reported to produce PHA. The phylogenetic analysis of the PHA synthase (phaC) gene of these bacteria showed a close homology with the phaC gene of different Pseudomonas species.This study indicates that stressed environments like oil-contaminated sites can be potential sources of medium-chain-length PHA producers.     

Response of the soil bacterial community to the addition of toluene and toluene-degrading bacteria

Changes in soil bacterial communities after toluene and/or toluene degrading bacteria were added were monitored by growth on various media, and by the culture-independent method of Reverse Sample Genome Probing (RSGP). A total of 397 isolates that were cultured from toluene-amended and non-amended soil were identified using fatty acid methyl ester (FAME) analysis. In 75% of the soil samples, gram-positive bacteria dominated, primarily Bacillus sp. and Cellulomonas sp. In contrast, RSGP revealed Proteobacteria (α, β, and γ subgroups) to be the dominant species, with Bacillus sp. dominant in only one soil sample.In toluene-treated soil, FAME and RSGP analyses indicated that by day 5 the major bacterial populations were gram-negative bacteria, in particular, Pseudomonas sp., Acinetobacter sp., and Alcaligenes sp. When toluene and Pseudomonas putida D8 were coincidentally introduced, the proportion of Pseudomonas sp. in the bacterial population recovered using non-selective medium increased from 16 to 62% and then rapidly decreased to about 9%. When we used selective medium to monitor the population of P. putida D8, a rapid initial increase followed by a gradual decline was also observed. In samples analyzed by RSGP, D8 was one of the major species of the bacterial community at day 2, but its signal intensity dropped to 9.5% by day 5.The influence of D8 addition on the bacterial profile was monitored in growth-based examinations. Bacillus sp. and Pseudomonas sp. were initially dominant. By day 5, Bacillus sp. decreased while the Proteobacteria, (including Acinetobacter sp., Agrobacterium sp., Alcaligenes sp., Burkholderia sp., Erwinia sp., and Pseudomonas sp.) increased. At the same time, D8 decreased to a level indistinguishable from background. Conversely, RSGP analysis revealed the population dominance of P. putida (including D8) and Rhizobium fredii at day 2. Populations shifted toward Agrobacterium tumefaciens, Bacillus subtilis, R. fredii, and D8 by day 5.P. putida D8 levels could be monitored using RSGP when cultivation failed. However, cultivation of Bacillus sp. was always successful, while the organism was only occasionally detected by RSGP. While cultivation and RSGP methods comparably detected the same major bacterial populations, the overall bacterial diversity was greater with RSGP than with growth-based testing.     

Microbial community development in the rhizosphere of apple trees at a replant disease site

Apple replant disease (ARD) is a complex syndrome that affects young trees in replanted orchard sites causing necrotic lesions on feeder roots, stunted tree growth and reduced cumulative yields. Use of ARD-tolerant rootstocks is an emerging control strategy. We studied the bacterial, fungal, and oomycetes populations in the rhizosphere of five rootstock cultivars (M.7, M.26, G.16, G.30 and CG.6210) planted into the old tree row or grass lanes of a previous orchard in Ithaca, NY, to better understand the role of rhizosphere microbial communities in the prevalence and control of ARD. The possible involvement of antagonistic Pseudomonas species, Pythium spp., Phytophthora spp. and rhizosphere cyanide concentrations in ARD were also examined. The rootstocks M.7, M.26 and G.16 were susceptible to ARD, while G.30 and CG.6210 were more tolerant. Tree growth on the rootstocks M.7, M.26 and G.16 was reduced by 10% when planted in the old tree rows, but this did not significantly reduce yields in the first fruiting year. The susceptible rootstocks, M.7 and M.26, supported higher densities of culturable rhizosphere fungi and bacteria than G.16, G.30 and CG.6210. Over 2 years, microbial densities were highest in July, lower in May and lowest in September. The composition of bacterial and fungal communities in the rhizosphere was highly variable and changed over seasons and years, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analyses. Initial differences in fungal rhizosphere communities between the two planting positions converged 2 years after the trees were replanted. In contrast, the bacterial rhizosphere community composition still differed significantly between the two planting positions 3 years after the orchard was replanted. The bacterial and fungal rhizosphere community compositions of susceptible rootstocks, M.7 and M.26, differed from those of the tolerant rootstocks, G.30 and CG.6210; G.16, differed from all the other rootstocks. The observed effects of rootstocks, planting positions and time on microbial community composition were small relative to the high variability observed overall. Pythium spp. and Phytophthora spp. infestations were high and similar for all rootstocks and planting positions. Neither potentially antagonistic Pseudomonas nor rhizosphere cyanide concentrations appeared to be involved in the ARD-complex at the studied site. Avoiding replanting into the old tree rows coupled with use of tolerant rootstocks appear to be the best strategies for reducing ARD in replanted orchards. Changes in rhizosphere microbial communities are among the many factors that contribute to improved tree growth when these management strategies are used.