Survival and cell culturability of biocontrol Pseudomonas fluorescens CHA0 in the rhizosphere of cucumber grown in two soils of contrasting fertility status

 The effect of cucumber roots on survival patterns of the biocontrol soil inoculant Pseudomonas fluorescens CHA0-Rif was assessed for 22 days in two non-sterile soils, using a combination of total immunofluorescence cell counts, Kogure's direct viable counts and colony counts on plates containing rifampicin. In Eschikon soil (high fertility status for cucumber), CHA0-Rif persisted as culturable cells in bulk soil and in the rhizosphere, but colony counts were lower than viable counts and total cell counts inside root tissues. The occurrence of viable but non-culturable (VBNC) cells inside root tissues (5 log cells g^–1 root) was unlikely to have resulted from the hydrogen peroxide treatment used to disinfect the root surface, as hydrogen peroxide caused the death of CHA0-Rif cells in vitro. In Siglistorf soil (low fertility status for cucumber), the inoculant was found mostly as non-culturable cells. Colony counts and viable counts of CHA0-Rif were similar, both in bulk soil and inside root tissues, whereas in the rhizosphere viable counts exceeded colony counts at the last two samplings (giving about 7 log VBNC cells g^–1). In conclusion, soil type had a significant influence on the occurrence of VBNC cells of CHA0-Rif, although these cells were found in root-associated habitats (i.e. rhizosphere and root tissues) and not in bulk soil. Received: 12 November 1999     

Crop rotation and soil temperature influence the community structure of Aspergillus flavus in soil

Aspergillus flavus, the most important cause of aflatoxin contamination, has two major morphotypes commonly termed ‘S’ and ‘L’ strains. Strain S isolates, on average, produce more aflatoxins than the strain L isolates. The S strain has been implicated as the primary causal agent of several contamination events in both North America and Africa. Strain S incidence and A. flavus propagules were quantified periodically in 11 agricultural fields in South Texas from spring 2001 through spring 2003. Both A. flavus populations and S strain incidence varied significantly among seasons, with warm seasons having higher average quantities of A. flavus (718 CFU g⁻¹) and higher incidences of the S strain (32.3%) than cold seasons (403 CFU g⁻¹ and 16.9% incidence). Previous crop influenced both the quantity of A. flavus and S strains incidence. Corn favors higher soil populations of A. flavus (1628 CFU g⁻¹) compared to cotton (374 CFU g⁻¹) and sorghum (237 CFU g⁻¹). In the agroecosystem of South Texas, both cotton (23.7%) and sorghum (23.5%) favored greater S strain incidence compared to corn (14.0%). Soil surface temperature greatly influenced fungal communities with propagule density decreasing when daily average soil temperature was either below 18 °C or above 30 °C, and the proportion of A. flavus belonging to the S strain increasing as soil temperature increased. The results suggest it may be possible to manipulate crop rotations in order to reduce aflatoxin severity, and that periods of increased soil temperature drive selection of the highly toxigenic S strain of A. flavus in warm climates.     

Rapid estimation of microbial biomass in grassland soils by ultra-violet absorbance

A reliable and simple technique for estimating soil microbial biomass (SMB) is essential if the role of microbes in many soil processes is to be quantified. Conventional techniques are notoriously time-consuming and unreproducible. A technique was investigated that uses the UV absorbance at 280 nm of 0.5 M K₂SO₄ extracts of fumigated and unfumigated soils to estimate the concentrations of carbon, nitrogen and phosphorus in the SMB. The procedure is based on the fact that compounds released after chloroform fumigation from lysed microbial cells absorb in the near UV region. Using 29 UK permanent grassland soils, with a wide range of organic matter (2.9–8.0%) and clay contents (22–68%), it was demonstrated that the increase in UV absorbance at 280 nm after soil fumigation was strongly correlated with the SMB C (r=0.92), SMB N (r=0.90) and SMB P (r=0.89), as determined by conventional methods. The soils contained a wide range of SMB C (412–3412 μg g⁻¹ dry soil), N (57–346 μg g⁻¹ dry soil) and P (31–239 μg g⁻¹ dry soil) concentrations. It was thus confirmed that the UV absorbance technique described was a rapid, simple, precise and relatively inexpensive method of estimating soil microbial biomass.     

Development of two culture media for mass cultivation of <Emphasis Type="Italic">Azospirillum</Emphasis> spp. and for production of inoculants to enhance plant growth

High yield culture medium is fundamental for production of inoculants for plant growth-promoting bacteria. Based on substitution of glucose in tryptone–yeast extract–glucose medium by Na-gluconate or glycerol, two new culture media were developed for mass cultivation of the commonly used plant growth-promoting bacterium Azospirillum sp. After 18 h of incubation, these modifications increased populations of different strains of Azospirillum (to ∼10¹¹ cells ml⁻¹ [single cell count] and ∼5 × 10⁹ CFU ml⁻¹ [plate count method]), significantly reduced generation time, and were also suitable for production of common synthetic inoculants.     

Survival and persistence of genetically modified Sinorhizobium meliloti in soil

Studies were conducted to evaluate the survival and persistence of Sinorhizobium meliloti 104A14 and two acid phosphatase-negative mutants in Kirkland (fine, mixed, thermic Udertic Paleustolls) silt loam soils with various fertility levels, and to assess the impact of inoculation on nodule occupancy and soil microbial community structure in the inoculated alfalfa (Medicago sativa L.) rhizosphere. Recovery of the inoculated strains was 100% (in the order of 10⁸ cells g⁻¹ soil) immediately following inoculation to soils, but decreased from 10⁸ cells g⁻¹ soil to undetectable levels in a nutrient-poor soil within 32 days. In a nutrient-rich soil, approximately 2–3% (4.7–7.43×10⁶ cells g⁻¹ soil) of the mutants and 23% (5.84×10⁷ cells g⁻¹ soil) of the wild-type inocula persisted for more than 64 days. Survivability and persistence of the wild-type S. meliloti were significantly greater than that of the genetically modified acid phosphatase negative mutants in all the soils tested. The persistence and nodule occupancy of the introduced S. meliloti in sterile and non-sterile soils were also tested for two repeated alfalfa growth periods in the same plant growth units, with a 1 month interval in between and no additional inoculation for the second period. Nodule occupancy of the introduced S. meliloti in non-sterile soils ranged from 30 to 60% for the first period and 85 to 100% for the second period. Our results suggest that survival and persistence of S. meliloti was enhanced by alfalfa cultivation and increased soil fertility, but impaired by mutation of acid phosphatase genes regardless of phosphorus nutritional levels. Moreover, inoculation with genetically modified S. meliloti strain 104A14 promoted indigenous bacterial growth in soil (increased bacterial population from 1.4×10⁶ to 4.3×10⁶ cells g⁻¹ soil), but not the growth of fungi and yeast. However, inoculation of the wild-type S. meliloti or genetically modified mutants did not result in significant changes in microbial community structure as indicated by EP indices and ratios of r/K strategists.     

Wood biochar increases nitrogen retention in field settings mainly through abiotic processes

Nitrogen (N) is an essential element associated with crop yield and its availability is largely controlled by microbially-mediated processes. The abundance of microbial functional genes (MFG) involved in N transformations can be influenced by agricultural practices and soil amendments. Biochar may alter microbial functional gene abundances through changing soil properties, thereby affecting N cycling and its availability to crops. The objective of this study was to assess the effects of wood biochar application on N retention and MFG under field settings. This was achieved by characterising soil labile N and their stable isotope compositions and by quantifying the gene abundance of nifH (nitrogen fixation), narG (nitrate reduction), nirS, nirK (nitrite reduction), nosZ (nitrous oxide reduction), and bacterial and archeal amoA (ammonia oxidation). A wood-based biochar was applied to a macadamia orchard soil at rates of 10 t ha⁻¹ (B10) and 30 t ha⁻¹ (B30). The soil was sampled after 6 and 12 months. The abundance of narG in both B10 and B30 was lower than that of control at both sampling months. Canonical Correspondence Analysis showed that soil variables (including dissolved organic C, NO₃⁻–N and NH₄⁺–N) and sampling time influenced MFG, but biochar did not directly impact on MFG. Twelve months after biochar application, NH₄⁺–N concentrations had significantly decreased in both B10 (4.74 μg g⁻¹) and B30 (5.49 μg g⁻¹) compared to C10 (13.9 μg g⁻¹) and C30 (17.9 μg g⁻¹), whereas NO₃⁻–N concentrations increased significantly in B30 (24.7 μg g⁻¹) compared to B10 (12.7 μg g⁻¹) and control plots (6.18 μg g⁻¹ and 7.97 μg g⁻¹ in C10 and C30 respectively). At month 12, significant δ¹⁵N of NO₃⁻–N depletion observed in B30 may have been caused by a marked increase in NO₃⁻–N availability and retention in those plots. Hence, it is probable that the N retention in high rate biochar plots was mediated primarily by abiotic factors.     

Soil microbial biomass and organic matter fractions during transition from conventional to organic farming systems

The aim of this study was to investigate the response of soil microbial biomass and organic matter fractions during the transition from conventional to organic farming in a tropical soil. Soil samples were collected from three different plots planted with Malpighia glaba: conventional plot with 10 years (CON); transitional plot with 2 years under organic farming system (TRA); organic plot with 5 years under organic farming system (ORG). A plot under native vegetation (NV) was used as a reference. Soil microbial biomass C (MBC) and N (MBN), soil organic carbon (SOC) and total N (TN), soil organic matter fractioning and microbial indices were evaluated in soil samples collected at 0–5, 5–10, 10–20 and 20–40 cm depth. SOC and fulvic acids fraction contents were higher in the ORG system at 0–5 cm and 5–10 cm depths. Soil MBC was highest in the ORG, in all depths, than in others plots. Soil MBN was similar between ORG, TRA and NV in the surface layer. The lowest values for soil MBC and MBN were observed in CON plot. Soil microbial biomass increased gradually from conventional to organic farming, leading to consistent and distinct differences from the conventional control by the end of the second year.     

Formation and use of microbial residues after adding sugarcane sucrose to a heated soil devoid of soil organic matter

A 67-day incubation experiment was carried out with a soil initially devoid of any organic matter due to heating, which was amended with sugarcane sucrose (C₄-sucrose with a δ¹³C value of ?10.5‰), inorganic N and an inoculum for recolonisation and subsequently at day 33 with C₃-cellulose (δ¹³C value of ?23.4‰). In this soil, all organic matter is in the microbial biomass or in freshly formed residues, which makes it possible to analyse more clearly the role of microbial residues for decomposition of N-poor substrates. The average δ¹³C value over the whole incubation period was ?10.7‰ in soil total C in the treatments without C₃-cellulose addition. In the CO₂ evolved, the δ¹³C values decreased from ?13.4‰ to ?15.4‰ during incubation. In the microbial biomass, the δ¹³C values increased from ?11.5‰ to ?10.1‰ at days 33 and 38. At day 67, 36% of the C₄-sucrose was left in the treatment without a second amendment. The addition of C₃-cellulose resulted in a further 7% decrease, but 4% of the C₃-cellulose was lost during the second incubation period. Total microbial biomass C declined from 200 μg g^?1 soil at day 5 to 70 μg g^?1 soil at day 67. Fungal ergosterol increased to 1.5 μg g^?1 soil at day 12 and declined more or less linearly to 0.4 μg g^?1 soil at day 67. Bacterial muramic acid declined from a maximum of 35 μg g^?1 soil at day 5 to a constant level of around 16 μg g^?1 soil. Glucosamine showed a peak value at day 12. Galactosamine remained constant throughout the incubation. The fungal C/bacterial C ratio increased more or less linearly from 0.38 at day 5 to 1.1 at day 67 indicating a shift in the microbial community from bacteria to fungi during the incubation. The addition of C₃-cellulose led to a small increase in C₃-derived microbial biomass C, but to a strong increase in C₄-derived microbial biomass C. At days 45 and 67, the addition of N-free C₃-cellulose significantly decreased the C/N ratio of the microbial residues, suggesting that this fraction did not serve as an N-source, but as an energy source.     

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 10⁴ CFU equivalents g⁻¹ lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10⁴ CFU equivalents g⁻¹. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.     

Effects of tree identity dominate over tree diversity on the soil microbial community structure

This study investigated the possible effects of tree species diversity and identity on the soil microbial community in a species-rich temperate broad-leaved forest. For the first time, we separated the effects of tree identity and tree species diversity on the link between above and belowground communities in a near-natural forest. We established 100 tree clusters consisting of each three tree individuals represented by beech (Fagus sylvatica L.), ash (Fraxinus excelsior L.), hornbeam (Carpinus betulus L.), maple (Acer pseudoplatanus L.), or lime (Tilia spec.) at two different sites in the Hainich National Park (Thuringia, Germany). The tree clusters included one, two or three species forming a diversity gradient. We investigated the microbial community structure, using phospholipid fatty acid (PLFA) profiles, in mineral soil samples (0–10 cm) collected in the centre of each cluster.The lowest total PLFA amounts were found in the pure beech clusters (79.0 ± 23.5 nmol g⁻¹ soil dw), the highest PLFA amounts existed in the pure ash clusters (287.3 ± 211.3 nmol g⁻¹ soil dw). Using principle components analyses (PCA) and redundancy analyses (RDA), we found only for the variables ‘relative proportion of beech trees’ and ‘living lime fine root tips associated with ectomycorrhiza’ a significant effect on the PLFA composition. The microbial community structure was mainly determined by abiotic environmental parameters such as soil pH or clay content. The different species richness levels in the clusters did not significantly differ in their total PLFA amounts and their PLFA composition. We observed a tendency that the PLFA profiles of the microbial communities in more tree species-rich clusters were less influenced by individual PLFAs (more homogenous) than those from species-poor clusters.We concluded that tree species identity and site conditions were more important factors determining the soil microbial community structure than tree species diversity per se.     

Changes in microbial community structure in soil as a result of different amounts of nitrogen fertilization

The changes in size, activity and structure of soil microbial community caused by N fertilization were studied in a laboratory incubation experiment. The rates of N fertiliser applied (KNO₃) were 0 (control), 100 and 2,000 μg N g⁻¹ soil. Despite no extra C sources added, a high percentage of N was immobilized. Whereas no significant increase of microbial C was revealed during incubation period, microbial growth kinetics as determined by the substrate-induced growth-response method demonstrated a significant decrease in the specific growth rate of microbial community in soil treated with 2,000 μg N g⁻¹ soil. Additionally, a shift in microbial community structure resulting in an increase in fungal biomarkers, mainly in the treatment with 2,000 μg N g⁻¹ soil was visible.     

Microbial growth efficiencies across a soil moisture gradient assessed using C-acetic acid vapor and N-ammonia gas

One integrative measurement of microbial activity in soils is the efficiency by which microbes convert assimilated carbon (C) into biomass C. This efficiency, called the microbial growth efficiency (Y), is a key physiological characteristic that regulates soil carbon sequestration, nutrient immobilization, and greenhouse gas emissions. Changes in rainfall patterns and soil water content as the result of global climate change have the potential to influence microbial activity and lead to changes in Y and thus, nutrient cycling at the ecosystem level. Unfortunately, little information is available on how environmental variables such as soil moisture influence Y. We have developed a new method for injecting ¹³C-labeled carbon as acetic acid vapor into soil that will allow measurement of microbial growth efficiency (as Y_C) without increasing soil moisture content. We compare Y determined with this new approach with an alternate method where injected ¹⁵N-labeled ammonia gas is used to quantify microbial N immobilization, and microbial growth efficiency is calculated based on microbial C:N and respiration rate (as Y_N). We also include injections of a solution containing labeled ammonium and acetate in our experiment to compare the results of our vapor methods with more commonly employed liquid-based methods. The ¹³C-acetic acid vapor, which was supplied to soils with soil moisture content ranging from 0.05 to 0.21 g H₂O g⁻¹ soil, was readily assimilated and respired by microbes. Between 0.10 and 0.21 g H₂O g⁻¹ soil (−0.60 to −0.04 MPa), values of Y_C averaged 0.46, and were significantly lower than values of Y_N, with average values of 0.58. Over this range, soil moisture content had no significant effect on either Y_C or Y_N. However, at the lowest soil moisture content (0.05 g H₂O g⁻¹ soil; <−6.0 MPa), Y_C and Y_N diverged substantially, suggesting that in very dry soils, constraints on microbial growth cause differential uptake of C and N resources.     

Depth-specific distribution and importance of nitrite-dependent anaerobic ammonium and methane-oxidising bacteria in an urban wetland

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) are two recently discovered processes in the nitrogen cycle that are catalysed by anammox bacteria and n-damo bacteria, respectively. Here, the depth-specific distribution and importance of anammox bacteria and n-damo bacteria were studied in an urban wetland, Xixi Wetland, Zhejiang Province (China). Anammox bacteria related to Candidatus Brocadia, Candidatus Kuenenia and Candidatus Anammoxoglobus, and n-damo bacteria related to “Candidatus Methylomirabilis oxyfera” were present in the collected soil samples. The abundance of anammox bacteria (2.6–8.6 × 10⁶ copies g⁻¹ dry soil) in the shallow soils (0–10 cm and 20–30 cm) was higher than that (2.5–9.8 × 10⁵ copies g⁻¹ dry soil) in the deep soils, whereas the abundance of n-damo bacteria (0.6–1.3 × 10⁷ copies g⁻¹ dry soil) in the deep soils (50–60 cm and 90–100 cm) was higher than that (3.4–4.5 × 10⁶ copies g⁻¹ dry soil) in the shallow soils. Anammox activity was detected at all depths, and higher potential rates (12.1–21.4 nmol N₂ g⁻¹ dry soil d⁻¹) were observed at depths of 0–10 cm and 20–30 cm compared with the rates (3.5–8.7 nmol N₂ g⁻¹ dry soil d⁻¹) measured at depths of 50–60 and 90–100 cm. In contrast, n-damo was mainly occurred at depths of 50–60 cm and 90–100 cm with potential rates of 0.7–5.0 nmol CO₂ g⁻¹ dry soil d⁻¹. This study suggested the niche segregation of the anammox bacteria and n-damo bacteria in wetland soils, with anammox bacteria being active primarily in deep soils and n-damo bacteria being active primarily in shallow soils.     

High specific activity in low microbial biomass soils across a no-till evapotranspiration gradient in Colorado

The need to identify microbial community parameters that predict microbial activity is becoming more urgent, due to the desire to manage microbial communities for ecosystem services as well as the desire to incorporate microbial community parameters within ecosystem models. In dryland agroecosystems, microbial biomass C (MBC) can be increased by adopting alternative management strategies that increase crop residue retention, nutrient reserves, improve soil structure and result in greater water retention. Changes in MBC could subsequently affect microbial activities related to decomposition, C stabilization and sequestration. We hypothesized that MBC and potential microbial activities that broadly relate to decomposition (basal and substrate-induced respiration, N mineralization, and β-glucosidase and arylsulfatase enzyme activities) would be similarly affected by no-till, dryland winter wheat rotations distributed along a potential evapotranspiration (PET) gradient in eastern Colorado. Microbial biomass was smaller in March 2004 than in November 2003 (417 vs. 231 μg g⁻¹ soil), and consistently smaller in soils from the high PET soil (191 μg g⁻¹) than in the medium and low PET soils (379 and 398 μg g⁻¹, respectively). Among treatments, MBC was largest under perennial grass (398 μg g⁻¹). Potential microbial activities did not consistently follow the same trends as MBC, and the only activities significantly correlated with MBC were β-glucosidase (r = 0.61) and substrate-induced respiration (r = 0.27). In contrast to MBC, specific microbial activities (expressed on a per MBC basis) were greatest in the high PET soils. Specific but not total activities were correlated with microbial community structure, which was determined in a previous study. High specific activity in low biomass, high PET soils may be due to higher microbial maintenance requirements, as well as to the unique microbial community structure (lower bacterial-to-fungal fatty acid ratio and lower 17:0 cy-to-16:1ω7c stress ratio) associated with these soils. In conclusion, microbial biomass should not be utilized as the sole predictor of microbial activity when comparing soils with different community structures and levels of physiological stress, due to the influence of these factors on specific activity.     

Arable weeds,cover crops,and tillage drive soil microbial community composition in organic cropping systems

Cover crops have traditionally been used to reduce soil erosion and build soil quality, but more recently cover crops are being used as an effective tool in organic weed management. Many studies have demonstrated microbial community response to individual cover crop species, but the effects of mixed species cover crop communities have received less attention. Moreover, the relationship between arable weeds and soil microbial communities is not well understood. The objective of this study was to determine the relative influence of cover crop diversity, early-season weed communities, and tillage on soil microbial community structure in an organic cropping system through the extraction of fatty acid methyl esters (FAMEs). A field experiment was conducted between 2009 and 2011 near Mead, NE where spring-sown mixtures of zero (control), two, and eight cover crop species were included in a sunflower–soybean–corn crop rotation. A mixture of four weed species was planted in all experimental units (excluding the no-cover control), and also included as an individual treatment. Cover crops and weeds were planted in late-March, then terminated in late-May using a field disk or sweep plow undercutter, and main crops were planted within one week of termination. Three (2009) or four (2010–11) soil cores were taken to a depth of 20 cm in all experimental units at 45, 32, and 25 days following cover crop termination in 2009, 2010, and 2011, respectively. Total FAMEs pooled across 2009 and 2010 were greatest in the two species mixture–undercutter treatment combination (140.8 ± 3.9 nmol g⁻¹) followed by the eight species mixture–undercutter treatment combination (132.4 ± 3.9 nmol g⁻¹). Abundance of five (2009 and 2010) and seventeen (2011) FAME biomarkers was reduced in the weedy treatment relative to both cover-cropped treatments and the no-cover control. In 2009 and 2010, termination with the undercutter reduced abundance of most actinomycete biomarkers while termination with the field disk reduced abundance of C18:1(cis11) and iC16:0. Canonical discriminant analysis of the microbial community successfully segregated most cover crop mixture by termination method treatment combinations in 2009 and 2010. Microbial communities were most strongly influenced by the presence and type of early-spring plant communities, as weeds exerted a strong negative influence on abundance of many key microbial biomarkers, including the AMF markers C16:1(cis11) and C18:1(cis11). Weeds may alter soil microbial community structure as a means of increasing competitive success in arable soils, but this relationship requires further investigation.     

Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).     

Decomposition of N-labelled maize leaves in soil affected by endogeic geophagous Aporrectodea caliginosa

A microcosm experiment was carried out for 56 days at 12 °C to evaluate the feeding effects of the endogeic geophagous earthworm species Aporrectodea caliginosa on the microbial use of ¹⁵N-labelled maize leaves (Zea mays) added as 5 mm particles equivalent to 1 mg C and 57 μg N g⁻¹ soil. The dry weight of A. caliginosa biomass decreased in the no-maize treatment by 10% during the incubation and increased in the maize leaf treatments by 18%. Roughly 5% and 10% of the added maize leaf-C and leaf-N, respectively, were incorporated into the biomass of A. caliginosa. About 29% and 33% of the added maize leaf-C were mineralised to CO₂ in the no-earthworm and earthworm treatments, respectively. The presence of A. caliginosa significantly increased soil-derived CO₂ production by 90 μg g⁻¹ soil in the no-maize and maize leaf treatments, but increased the maize-derived CO₂ production only by 40 μg g⁻¹ soil. About 10.5% of maize leaf-C and leaf-N was incorporated into the soil microbial biomass in the absence of earthworms, but only 6% of the maize leaf-C and 3% of the maize leaf-N in the presence of earthworms. A. caliginosa preferentially fed on N rich, maize leaf-colonizing microorganisms to meet its N demand. This led to a significantly increased C/N ratio of the unconsumed microbial biomass in soil. The ergosterol-to-microbial biomass C ratio was not significantly decreased by the presence of earthworms. A. caliginosa did not directly contribute to comminution of plant residues, as indicated by the absence of any effects on the contents of the different particulate organic matter fractions, but mainly to grazing of residue-colonizing microorganisms, increasing their turnover considerably.     

Field survival of the phytostimulator Azospirillum lipoferum CRT1 and functional impact on maize crop, biodegradation of crop residues, and soil faunal indicators in a context of decreasing nitrogen fertilisation

Heavy nitrogen fertilisation is often implemented in maize cropping systems, but it can have negative environmental effects. Nitrogen-fixing, phytohormone-producing Azospirillum plant growth-promoting rhizobacteria (PGPR) have been proposed as crop inoculants to maintain high yield when decreasing nitrogen fertilisation. In this context, agronomic and ecological effects of the inoculation of maize seeds with the PGPR Azospirillum lipoferum CRT1 were studied in two consecutive years. The inoculant was recovered from maize at 10⁵ CFU g⁻¹ root or higher. Inoculation enhanced root growth and development based on results of root biomass, rooting depth and/or parameters describing root system architecture, and a transient positive effect on shoot height was observed in the first year. Inoculation did not increase yield, but reducing mineral nitrogen fertilisation had only a minor effect on yield. This suggests that the lack of positive effect of the PGPR on yield was due to the fact that the whole field was heavily fertilised in years prior to the start of the experiment. Soil nitrogen levels decreased during the 2 years of the study, and the inoculant had no effect on residual soil nitrogen levels at harvest. Inoculation had no impact on Fusarium symptoms and concentration of the mycotoxin deoxynivalenol in maize kernels, but both were influenced by the interaction between inoculation and nitrogen fertilisation level. Inoculation did not influence meso/macrofaunal soil populations, but had a small but significant effect (smaller than the effect of added nitrogen) on decomposition, nitrogen mineralisation and mesofaunal colonisation of maize leaves (in litter bags). Overall, the ecological impact of seed inoculation with the PGPR A. lipoferum CRT1 was small, and its magnitude was smaller than that of chemical nitrogen fertilisation.     

Immobilization and mineralization of nitrogen in a saline and alkaline soil during microbial use of sugarcane filter cake amended with glucose

A 42-day incubation was conducted to study the effect of glucose and ammonium addition adjusted to a C/N ratio of 12.5 on sugarcane filter cake decomposition and on the release of inorganic N from microbial residues formed initially. The CO₂ evolved increased in comparison with the non-amended control from 35% of the added C with pure +5 mg g⁻¹ soil filter cake amendment to 41% with +5 mg g⁻¹ soil filter cake +2.5 mg g⁻¹ soil glucose amendment to 48% with 5 mg g⁻¹ soil filter cake +5 mg g⁻¹ soil glucose amendment. The different amendments increased microbial biomass C and microbial biomass N within 6 h and such an increase persisted. The fungal cell-membrane component ergosterol initially showed a disproportionate increase in relation to microbial biomass C, which completely disappeared by the end of the incubation. The cellulase activity showed a 5-fold increase after filter cake addition, which was not further increased by the additional glucose amendment. The cellulase activity showed an exponential decline to values around 4% of the initial value in all treatments. The amount of inorganic N immobilized from day 0 to day 14 increased with increasing amount of C added, in contrast to the control treatment. After day 14, the immobilized N was re-mineralized at rates between 1.3 and 1.5 μg N g⁻¹ soil d⁻¹ in the treatments being more than twice as high as in the control treatment. This means that the re-mineralization rate is independent of the actual size of the microbial residues pool and also independent of the size of the soil microbial biomass.     

Soil bacterial functional diversity in a potato field

Nutrient availability of plants varies according to different processes governed by soil biota. In agroecosystems, human intervention may affect soil biota and therefore it has a crucial impact on system productivity and its maintenance. Based on the above information, we assumed that sequencing bacterial functional diversity in agrosystems will be affected by plant growing stages and human activity (agricultural practice). During the study period, soil samples comprising five cores (5 cm diameter) from upper (0 to 10 cm) and deeper (10 to 20 cm) layers were collected individually from a potato field and from a control site with zero input treatment. Soil moisture, total organic carbon and bacterial functional diversity were assessed. The results obtained from the field and laboratory studies demonstrate differences between growing stages. The percentage of soil moisture content ranged between 10–12 % during the study period, independent of depth, location (treatment and control) and growth stage, whereas total organic carbon (TOC) oscillated between 0.15–0.35 %. Soil microbial biomass (SMB) measured in the upper layer (0 to 10 cm) increased from values of 100 μg C·g^–1 soil before planting to 190 μg C·g^–1 soil after yield harvesting, and in the deep soil layer (10 to 20 cm) a mean value of 80 μg C·g^–1 soil was obtained. Bacterial functional diversity (BFD) was evaluated using the Biolog method. Values of Shannon diversity H’ = 16·10^–2 obtained in the upper layer during the pre-planting stage decreased to H’ = 5·10^–2 after planting. At the deep layer (10 to 20 cm), similar trends to those measured in the upper layer (0 to 10 cm) were obtained, except during the harvesting and post-harvesting seasons, when maximal values of H’ = 30·10^–2 were detected. In this context, we tried to comprehend the dynamics of microbial community and the diversity of bacterial populations participating in key soil processes of agroecosystems.