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.     

Impact of inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the genetic structure of the rhizobacterial community of field-grown maize

The phytostimulatory PGPR Azospirillum lipoferum CRT1 was inoculated to maize seeds and the impact on the genetic structure of the rhizobacterial community in the field was determined during maize growth by Automated Ribosomal Intergenic Spacer Analysis (ARISA) of rhizosphere DNA extracts. ARISA fingerprints could differ from one plant to the next as well as from one sampling to the next. Inoculation with strain CRT1 enhanced plant-to-plant variability of the ARISA fingerprints and caused a statistically significant shift in the composition of the indigenous rhizobacterial community at the first two samplings. This is the first study on the ecological impact of Azospirillum inoculation on resident bacteria done in the field and showing that this impact can last at least one month.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Rapid quantification of Bacillus subtilis antibiotics in the rhizosphere

Biological control agents like Bacillus subtilis offer an alternative and supplement to synthetic pesticides. Antibiotic production by biocontrol strains of B. subtilis can play a major role in plant disease suppression. Our current understanding of B. subtilis antibiosis comes from culture media measurements of antibiotic production and in vitro suppression of pathogens. Quantifying the antibiotic metabolite chemistry of B. subtilis biofilms growing on root surfaces provides a more accurate understanding of in vivo antibiotic production. An analytical method based on solid-phase extraction (SPE) with high-performance liquid chromatography (HPLC) and mass spectroscopy (MS) has been developed to quantify antibiotics produced by B. subtilis growing on plant roots. Cucumber (Cucumis sativus) was grown in composted soil and potting media inoculated with B. subtilis strain QST 713 (AgraQuest, USA). Two important B. subtilis antibiotics, surfactin and iturin A, were extracted from root and rhizosphere soil using acidified organic solvents followed by cleaning and concentration using SPE. HPLC and HPLC-MS were used to measure surfactin and iturin A. Rhizosphere concentrations of both antibiotics increased with plant age. For plants grown in peat-based potting media, surfactin concentrations increased from 9 μg g⁻¹ root fresh weight (RFW) at 15 d to 30 μg g⁻¹ RFW at 43 d. Iturin concentrations were 7 μg g⁻¹ RFW at 15 d and 180 μg g⁻¹ RFW at 43 d. In an initial field trial in a composted fine sandy loam, we demonstrated rhizosphere production of surfactin and iturin under competition and predation by the myriad macro- and microfauna existing in a fertile high-organic soil, with mature B. subtilis-inoculated cucumber roots yielding 33 μg g⁻¹ RFW surfactin and 630 μg g⁻¹ RFW iturin at 78 d.     

Pseudomonas corrugata: A suitable bacterial inoculant for maize grown under rainfed conditions of Himalayan region

Colonization and survival of the inoculated bacteria in rhizosphere of maize were investigated in field and pot experiments conducted for 3 consecutive years under rainfed conditions of Himalayan region. The effect of bacterial inoculations on growth and yield related parameters of maize were also evaluated. While three bacterial species, viz. Bacillus megaterium, Bacillus subtilis and Pseudomonas corrugata were tested in 1st year experiments, P. corrugata (based on the 1st year results) was chosen for inoculation in the subsequent experiments. All the three bacterial inoculants showed good rhizosphere competence giving high inoculum numbers (log₁₀ 11.13-11.34 cfu g⁻¹). The bacterial inoculations by B. megaterium, B. subtilis and P. corrugata resulted in an increment in grain yield of maize up to 122.4%, 135.2% and 194.3%, respectively, as compared to respective control. In 1st year, the antibiotic marked (Nal^r Rif^r) inoculant P. corrugata resulted in the highest increase in grain yield, statistically significant (P<0.05) as compared to control, B. megaterium and B. subtilis. In 2nd and 3rd year experiments, P. corrugata increased the grain yield up to 147.28% and 149.93%, respectively, as compared to control. The best performance and consistent trend of P. corrugata to increase plant yields was credited to its initial isolation from rhizosphere of maize growing under temperate conditions. The overall beneficial effects of bacterial inoculations on maize were contributed to (1) the colonization and survival of the introduced bacteria, and (2) stimulation of the indigenous microflora in the rhizosphere. Based on the comprehensive results obtained in this study, P. corrugata may be recommended as suitable bioinoculant for maize fields of temperate climate grown under rainfed conditions.     

Release of the recombinant Cry3Bb1 protein of Bt maize MON88017 into field soil and detection of effects on the diversity of rhizosphere bacteria

A three-year experimental field study with a genetically engineered Bt maize (event MON88017) and three conventionally bred cultivars was conducted to quantify the recombinant Cry3Bb1 protein released into soil and detect effects on the diversity of soil bacteria. Protein extraction and an enzyme-linked immunosorbent assay (ELISA) allowed a threshold detection of 0.01 ng Cry3Bb1 g^?1 soil. The maximum amount found in field plots with Bt maize was 1.0 ng Cry3Bb1 g^?1 rhizosphere soil. Average concentrations during the growing seasons varied between years from 0.07 to 0.29 ng g^?1. No accumulation of Cry3Bb1 in soil occurred over the three growing seasons. Four weeks after harvest, the major Cry3Bb1 reservoirs on the field were the remaining root stubbles, but their Cry3Bb1 concentration declined by 98.30–99.99% in the following seven months. During the three consecutive years of study there were never significant differences between the rhizosphere bacterial community structure of the Bt maize and the other cultivars, as detected by cultivation independent profiling of PCR-amplified 16S rRNA genes. The low concentrations of soil extractable Cry3Bb1, its degradation in decaying roots, and the lack of effects on rhizosphere bacteria give no indications of adverse effects of MON88017 cultivation on soil ecology.     

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.     

Rhizosphere isoflavones (daidzein and genistein) levels and their relation to the microbial community structure of mono-cropped soybean soil in field and controlled conditions

Despite an increase in the understanding of the soybean isoflavones involved in root-colonizing symbioses, relatively little is known about their levels in the rhizosphere and their interactions with the soil microbial community. Based on a 13-year experiment of continuous soybean monocultures, in the present study we quantified isoflavones in the soybean rhizosphere and analyzed the soil microbial community structure by examining its phospholipid fatty acid (PLFA) profile. Two isoflavones, daidzein (7, 4′-dihydroxyisoflavone) and genistein (5,7,4′- trihydroxyisoflavone), were detected in the rhizosphere soil of soybean plants, with the concentrations in the field varying with duration of mono-cropping. Genistein concentrations ranged from 0.4 to 1.2 μg g⁻¹ dry soil over different years, while daidzein concentrations rarely exceeded 0.6 μg g⁻¹ dry soil. PLFA profiling showed that the signature lipid biomarkers of bacteria and fungi varied throughout the years of the study, particularly in mono-cropping year 2, and mono-cropping years 6-8. Principal component analysis clearly identified differences in the composition of PLFA during different years under mono-cropping. There was a positive correlation between the daidzein concentrations and soil fungi, whereas the genistein concentration showed a correlation with the total PLFA, fungi, bacteria, Gram (+) bacteria and aerobic bacteria in the soil microbial community. Both isoflavones were easily degraded in soil, resulting in short half-lives. Concentrations as small as 1 μg g⁻¹ dry soil were sufficient to elicit changes in microbial community structure. A discriminant analysis of PLFA patterns showed that changes in microbial community structures were induced by both the addition of daidzein or genistein and incubation time. We conclude that daidzein and genistein released into the soybean rhizosphere may act as allelochemicals in the interactions between root and soil microbial community in a long-term mono-cropped soybean field.     

Detection and quantification of the nematophagous fungus Hirsutella minnesotensis in soil with real-time PCR

A real-time PCR assay was developed to quantify in soil the fungus Hirsutella minnesotensis, an important parasite of secondary-stage juvenile (J2) of the soybean cyst nematode. A primer pair 5′-GGGAGGCCCGGTGGA-3′ and 5′-TGATCCGAGGTCAACTTCTGAA-3′ and a TaqMan probe 5′-CGTCCGCCGTAAAACGCCCAAC-3′ were designed based on the sequence of the ITS region of the rRNA gene. The primers were highly species-specific. The PCR reaction system was very sensitive and able to detect as few as 4 conidia g^?1 soil. Regression analysis showed similar slopes and efficiency on DNA from pure culture (y = ?3.587x + 41.017, R² = 0.9971, E = 0.9055) and from Log conidia g^?1 soil (y = ?3.855x + 37.669, R² = 0.9139, E = 0.8172), indicating that the real-time PCR protocol can reliably quantify H. minnesotensis in the soil. The real-time PCR assay was applied to 20 soil samples from soybean fields, and compared with a parasitism assay. The real-time PCR assay detected H. minnesotensis in six of the soils, whereas the parasitism assay detected H. minnesotensis in the same six soils and three additional soils. The real-time PCR assay was weakly correlated (R² = 0.49) with the percentage of parasitized J2 in the six soils, indicating that different types of soil may interfere the efficiency of the real-time PCR assay, possibly due to the effect of soil types on efficacy of DNA extraction. The parasitism assay appeared to be more sensitive than real-time PCR in detecting presence of H. minnesotensis, but real-time PCR was much faster and less costly and provided a direct assessment of fungal biomass. Using the two assays in combination can obtain more complete information about the fungus in soil than either assay alone. Hirsutella parasitism was widespread and detected in 13 of the 20 field soils, indicating that these fungi may contribute to suppressiveness of soybean cyst nematode in nature and likely have high biological control potential for the nematode.     

Impacts of different N management regimes on nitrifier and denitrifier communities and N cycling in soil microenvironments

Real-time quantitative PCR assays, targeting part of the ammonia monooxygenase (amoA), nitrous oxide reductase (nosZ), and 16S rRNA genes were coupled with ¹⁵N pool dilution techniques to investigate the effects of long-term agricultural management practices on potential gross N mineralization and nitrification rates, as well as ammonia-oxidizing bacteria (AOB), denitrifier, and total bacterial community sizes within different soil microenvironments. Three soil microenvironments [coarse particulate organic matter (cPOM; >250 μm), microaggregate (53-250 μm), and silt-and-clay fraction (<53 μm)] were physically isolated from soil samples collected across the cropping season from conventional, low-input, and organic maize-tomato systems (Zea mays L.-Lycopersicum esculentum L.). We hypothesized that (i) the higher N inputs and soil N content of the organic system foster larger AOB and denitrifier communities than in the conventional and low-input systems, (ii) differences in potential gross N mineralization and nitrification rates across the systems correspond with AOB and denitrifier abundances, and (iii) amoA, nosZ, and 16S rRNA gene abundances are higher in the microaggregates than in the cPOM and silt-and-clay microenvironments. Despite 13 years of different soil management and greater soil C and N content in the organic compared to the conventional and low-input systems, total bacterial communities within the whole soil were similar in size across the three systems (∼5.15 × 10⁸ copies g⁻¹ soil). However, amoA gene densities were ∼2 times higher in the organic (1.75 × 10⁸ copies g⁻¹ soil) than the other systems at the start of the season and nosZ gene abundances were ∼2 times greater in the conventional (7.65 × 10⁷ copies g⁻¹ soil) than in the other systems by the end of the season. Because organic management did not consistently lead to larger AOB and denitrifier communities than the other two systems, our first hypothesis was not corroborated. Our second hypothesis was also not corroborated because canonical correspondence analyses revealed that AOB and denitrifier abundances were decoupled from potential gross N mineralization and nitrification rates and from inorganic N concentrations. Our third hypothesis was supported by the overall larger nitrifier, denitrifier, and total bacterial communities measured in the soil microaggregates compared to the cPOM and silt-and-clay. These results suggest that the microaggregates are microenvironments that preferentially stabilize C, and concomitantly promote the growth of nitrifier and denitrifier communities, thereby serving as potential hotspots for N₂O losses.     

Distribution of Aspergillus section Flavi in soils of maize fields in three agroecological zones of Nigeria

Fungal communities in soils of Nigerian maize fields were examined to determine distributions of aflatoxin-producing fungi and to identify endemic atoxigenic strains of potential value as biological control agents for limiting aflatoxin contamination in West African crops. Over 1000 isolates belonging to Aspergillus section Flavi were collected from soil of 55 Nigerian maize fields located in three agroecological zones by dilution plating onto modified Rose Bengal agar. The most common member of Aspergillus section Flavi (85% of isolates) was the A. flavus L-strain followed by the unnamed taxon known as strain S_BG (8%), A. tamarii (6%) and A. parasiticus (1%). Highest incidence of S_BG was in Zaria district, and lowest was in Ogbomosho and Ado-Ekiti districts. Only 44% of 492 A. flavus isolates produced aflatoxins in liquid fermentation (limit of detection 5 ng g⁻¹). Thirty-two percent of the A. flavus isolates produced >1 μg g⁻¹ total aflatoxins but no A. flavus isolate produced G aflatoxins. When the agroecological zones were compared, significantly (P < 0.05) greater proportions of aflatoxigenic A. flavus isolates were found in the Northern Guinea Savannah (61%) than in Southern Guinea Savannah (31%). The Derived Savannah was intermediate between the other two agroecological zones. Each of the regions had atoxigenic strains of potential value as biological control agents. All S_BG and A. parasiticus isolates produced both B and G aflatoxins and greater than 300 μg g⁻¹ total aflatoxins. S_BG and A. parasiticus isolates were the greatest contributors to the aflatoxin-producing potential of fungal communities in regions where these isolates occurred.     

Volatile organic compounds in the roots and rhizosphere of Pinus spp.

Plant roots normally release a complex mixture of chemicals which have important effects in the rhizosphere. Among these different root-emitted compounds, volatile isoprenoids have received very little attention, yet they may play important and diverse roles in the rhizosphere, contributing to the regulation of microbial activity and nutrient availability. It is therefore important to estimate their abundance in the rhizosphere, but so far, there is no reliable sampling method that can be used to measure realistic rates of root emissions from plants growing in field conditions, or even in pots. Here, we measured root content of volatile isoprenoids (specifically monoterpenes) for Pinus pinea, and explored the feasibility of using a dynamic bag enclosure method to measure emissions from roots of intact pot-grown plants with different degrees of root cleaning. We also investigated a passive diffusion method for exploring monoterpenes in soil at incremental distances from mature Pinus sylvestris trees growing in field conditions. Total monoterpene content of P. pinea roots was 415±50 μg g⁻¹ fresh wt in an initial screening study, and between 688±103 and 1144±208 μg g⁻¹ dry wt in subsequent investigations. Emissions from shaken-clean roots of intact plants and roots of intact plants washed to remove remaining soil after shaken-clean experiments were 119±14 and 26±5 μg g⁻¹ dry wt h⁻¹, respectively. Emissions from intact roots in soil-balls were an order of magnitude lower than from shaken-clean roots, and probably reflected the amount of emitted compounds taken up by physical, chemical or biological processes in the soil matrix surrounding the roots. Although monoterpene content was not significantly different in droughted roots, emission rates from droughted roots were generally significantly lower than from well-watered roots. Finally, passive sampling of monoterpenes in the soil at different distances from mature P. sylvestris trees in field conditions showed significantly decreasing sampling rates with increasing distance from the trunk. We conclude that it is feasible to measure volatile isoprenoid emissions from roots but the method of root preparation affects magnitude of measured emissions and therefore must be decided according to the application. We also conclude that the rhizosphere of Pinus species is a strong and previously un-characterized source of volatile isoprenoid emissions and these are likely to impact significantly on rhizosphere function.     

Localisation of nitrate in the rhizosphere of biochar-amended soils

A wheat seedling rhizobox approach was used to differentiate between the rhizosphere and non-rhizosphere (bulk) soil amended with low and high rates of biochar (20 and 60 t ha⁻¹ vs. control). Nitrate (NO₃⁻) was added as the main nitrogen (N) source because emerging biochar research points to reduced NO₃⁻ loss through leaching and gaseous loss as nitrous oxide. The rhizosphere under the different treatments were distinct (P = 0.021), with greater soil-NO₃⁻ and biochar-NO₃⁻ contents in the high biochar treatment. Biochar addition increased wheat root length ratio (P = 0.053) and lowered root N uptake (P = 0.017), yet plant biomass and N content were similar between treatments. The results indicate localisation of NO₃⁻ within the rhizosphere of biochar-amended soils which has implications for NO₃⁻ loss and improved nitrogen use efficiency.     

Spatial distribution of arbuscular mycorrhizal fungi, glomalin and soil enzymes under the canopy of Astragalus adsurgens Pall. in the Mu Us sandland, China

To measure and manage plant growth in arid and semi-arid sandlands, improved understanding of the spatial patterns of desert soil resources and the role of arbuscular mycorrhizal (AM) fungi is needed. Spatial patterns of AM fungi, glomalin and soil enzyme activities were investigated in five plots located in the Mu Us sandland, northwestern China. Soils to 50 cm depth in the rhizosphere of Astragalus adsurgens Pall. were sampled. The study demonstrated that A. adsurgens Pall. could form strong symbiotic relationships with AM fungi. Arbuscular mycorrhizal fungal status and distributions were significantly different among the five studied plots. Correlation coefficient analysis demonstrated that spore density was significantly and positively correlated with soil organic carbon (SOC), soil acid phosphatase and to two Bradford-reactive soil protein (BRSP) fractions (P < 0.01). Colonization of arbuscules and vesicles were positively correlated with protease activity. The BRSP fractions were also significantly and positively correlated to edaphic factors (e.g. SOC, available nitrogen, and Olsen phosphorus) and soil enzymes (e.g. soil urease and acid phosphatase). The means of total BRSP and easily extractable BRSP were 0.95 mg g⁻¹ and 0.5 mg g⁻¹ in all data, respectively. The levels of BRSP in the desert soil were little lower than those in native and arable soils, but the ratios of BRSP to SOC were much higher than farmland soils. The results of this study support the conclusion that glomalin could be an appropriate index related to the level of soil fertility, especially in desert soil. Moreover, AM fungal colonizations and glomalin might be useful to monitor desertification and soil degradation.     

Humic acids buffer the effects of urea on soil ammonia oxidizers and potential nitrification

Humic acids (HAs) play an important role in the global nitrogen cycle by influencing the distribution, bioavailability, and ultimate fate of organic nitrogen. Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in ecosystems and is limited, in part, by the availability of NH₄⁺. We evaluated the impact of HAs on soil AOB in microcosms by applying urea (1.0%, equal to 10 mg urea/g soil) with 0.1% bHA (biodegraded lignite humic acids, equal to 1 mg/g soil), 0.1% cHA (crude lignite humic acids) or no amendment. AOB population size, ammonium and nitrate concentrations were monitored for 12 weeks after urea and HA application. AOB densities (quantified by real-time PCR targeting the amoA) in the Urea treatments increased about ten-fold (the final abundance: 5.02 × 10⁷ copies (g of dry soil)⁻¹) after one week of incubation and decreased to the initial density after 12 weeks incubation; the population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) decreased from 1.12 × 10¹⁰ to 2.59 × 10⁹ copies (g of dry soil)⁻¹ at week one and fluctuated back to the initial copy number at week 12. In the Urea + bHA and Urea + cHA treatments, the AOB densities were 4 and 6 times higher, respectively, than the initial density of approximately 5.07 × 10⁶ copies (g of dry soil)⁻¹ at week 1 and did not change much up to week 4; the total bacteria density changed little over time. The AOB and total bacteria density of the controls changed little during the 12 weeks of incubation. The microbial community composition of the Urea treatment, based on T-RFLP using CCA (canonical correspondence analysis) and pCCA (partial CCA) analysis, was clearly different from those of other treatments, and suggested that lignite HAs buffered the change in diversity and quantity of total bacteria caused by the application of urea to the soil. We hypothesize that HAs can inhibit the change in microbial community composition and numbers, as well as AOB population size by reducing the hydrolysis rate from urea to ammonium in soils amended with urea.     

Respiration pattern and microbial use of field-grown transgenic Bt-maize residues

A 49-day incubation experiment was carried out with the addition of field-grown maize stem and leaf residues to soil at three different temperatures (5, 15, and 25 °C). The aim was to study the effects of two transgenic Bt-maize varieties in comparison to their two parental non-Bt varieties on the mineralization of the residues, on their incorporation into the microbial biomass and on changes in the microbial community structure. The stem and leaf residues of Novelis-Bt contained 3.9 μg g⁻¹ dry weight of the Bt toxin Cry1Ab and those of Valmont-Bt only 0.8 μg g⁻¹. The residues of the two parental non-Bt varieties Nobilis and Prelude contained higher concentrations of ergosterol (+220%) and glucosamine (+190%) and had a larger fungal C-to-bacterial C ratio (+240%) than the two Bt varieties. After adding the Bt residues, an initial peak in respiration of an extra 700 μg CO₂-C g⁻¹ soil or 4% of the added amount was observed in comparison to the two non-Bt varieties at all three temperatures. On average of the four varieties, 19-38% of the maize C added was mineralized during the 49-day incubation at the three different temperatures. The overall mean increase in total maize-derived CO₂ evolution corresponded to a Q₁₀ value of 1.4 for both temperature steps, i.e. from 5 to 15 °C and from 15 to 25 °C. The addition of maize residues led to a strong increase in all microbial properties analyzed. The highest contents were always measured at 5 °C and the lowest at 25 °C. The variety-specific contents of microbial biomass C, biomass N, ATP and adenylates increased in the order Novelis-Bt ? Prelude<Valmont-Bt ? Nobilis. The mineralization of Novelis-Bt residues with the highest Bt concentration and lowest N concentration and their incorporation into the microbial biomass was significantly reduced compared to the parental non-Bt variety Nobilis. These negative effects increased considerably from 5 to 25 °C. The transgenic Bt variety Valmont did not show further significant effects except for the initial peak in respiration at any temperature.     

DIBOA: Fate in soil and effects on root-knot nematode egg numbers

The benzoxazinoid 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) is produced by rye (Secale cereale) and may contribute to plant-parasitic nematode suppression when rye plants are incorporated as a green manure. We investigated the fate of DIBOA in soil and DIBOA's effects on nematode reproduction. Soil in plastic bags was treated with DIBOA at concentrations ranging from 1.1 to 18 μg g⁻¹ dry soil, and with the root-knot nematode Meloidogyne incognita. Control soils were treated with water or with 0.31% methanol, with or without nematodes. DIBOA concentrations extracted from the soil were measured at selected times for 5 consecutive days. The soil from each bag was then placed into a pot in the greenhouse, and a cucumber seedling was transplanted into each pot. Five weeks later, only the highest DIBOA concentration, 18 μg g⁻¹ soil, reduced nematode egg numbers. At 0 h, DIBOA measured in soil ranged from 19.68 to 35.51% of the initial DIBOA concentration, and was dependent on the concentration added to the soil. DIBOA half-life was from 18 to 22 h, and very little DIBOA was present in soil after 120 h. Identified breakdown products accounted for only 4% at maximum of the initially added DIBOA. The results of our study demonstrate that high soil concentrations of DIBOA are necessary to suppress M. incognita; DIBOA may not be a major factor in nematode suppression by a rye cover crop.     

Identification and quantification of Fusarium oxysporum in planta and soil by means of an improved specific and quantitative PCR assay

The proper identification and quantification of F. oxysporum populations inhabiting soil and plant rhizosphere niches are of importance for soil microbial ecology and plant pathology. In this study, we report the improvement of a PCR protocol for the specific identification of the F. oxysporum species complex and its conversion into a real-time qPCR assay for the quantification up to 1 pg of the fungus DNA in soil and different plant tissues. The amplification efficiency, sensibility and reproducibility of qPCR assays were not influenced by presence of non-target DNA from either plant or soil. The applicability of the newly developed qPCR protocol for F. oxysporum population studies was demonstrated using the technique for quantifying the fungus in different complex environmental samples. The use of the qPCR protocol allowed to accurately quantify up to 25 pg of F. oxysporum/g of naturally infested field soil, as well as to identify significant differences in the amount of F. oxysporum DNA in roots of different chickpea cultivars grown in a field soil infested with diverse pathogenic and nonpathogenic F. oxysporum populations. This qPCR protocol may be especially important for studies on soil microbial ecology and plant pathology since it provides a new opportunity for analyzing F. oxysporum populations and their interactions with the soil microflora, environment and plant host genotypes.     

Phytase and phosphatase producing fungi in arid and semi-arid soils and their efficiency in hydrolyzing different organic P compounds

Seven most efficient phytase and phosphatases producing fungi were isolated from the soils of arid and semi-arid regions of India and tested for their efficiency on hydrolysis of two important organic P compounds: phytin and glycerophosphate. The native soil organic P may be exploited after using these organisms as seed inoculants, to help attain higher P nutrition of plants. The identified organisms belong to the three genera: Aspergillus, Emmericella and Penicillium. Penicillium rubrum released the most acid into the medium during growth. Aspergillus niger isolates were found to accumulate biomass the fastest. A significant negative correlation (r=−0.593,n=21, p<0.01) was observed between the development of fungal mat and pH of the media. The extracellular (E) phosphatases released by different fungi were less than their intracellular (I) counterpart, but the trend was reversed in case of phytase production. The E:I ratio of different fungi ranged from 0.39 to 0.86 for acid phosphatase, 0.29 to 0.41 for alkaline phosphatases and 9.4 to 19.9 for phytase. The efficiency of hydrolysis of different organic P compounds of different fungi varied from 2.12-4.85 μg min⁻¹ g⁻¹ for glycerophosphate to 0.92-2.10 μg min⁻¹ g⁻¹ for phytin. The trend of efficiency was as follows: Aspergillus sp.>Emmericella sp.>Penicillium sp. The results indicated that the identified fungi have enough potential to exploit native organic phosphorus to benefit plant nutrition.