Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Strong temperature dependence and no moss photosynthesis in winter CO2 flux for a Kobresia meadow on the Qinghai-Tibetan plateau

We examined the CO₂ exchange of a Kobresia meadow ecosystem on the Qinghai-Tibetan plateau using a chamber system. CO₂ efflux from the ecosystem was strongly dependence on soil surface temperature. The CO₂ efflux-temperature relationship was identical under both light and dark conditions, indicating that no photosynthesis could be detected under light conditions during the measurement period. The temperature sensitivity (Q₁₀) of the CO₂ efflux showed a marked transition around −1.0 °C; Q₁₀ was 2.14 at soil surface temperatures above and equal to −1.0 °C but was 15.3 at temperatures below −1.0 °C. Our findings suggest that soil surface temperature was the major factor controlling winter CO₂ flux for the alpine meadow ecosystem and that freeze-thaw cycles at the soil surface layer play an important role in the temperature dependence of winter CO₂ flux.     

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.     

Evaluation of different Coniothyrium minitans inoculum sources and application rates on apothecial production and infection of Sclerotinia sclerotiorum sclerotia

The effects of three Coniothyrium minitans isolates (Conio, IVT1 and Contans^®), applied to soil as conidial suspensions or as maizemeal-perlite (MP) inocula (Conio), on apothecial production and infection of Sclerotinia sclerotiorum sclerotia were assessed in two soil pot bioassays and two novel box bioassays in the glasshouse at different times of the year. C. minitans isolate Conio applied as either MP or ground MP at full rate (10⁶-10⁷ cfu cm⁻³ soil) consistently decreased the carpogenic germination, recovery and viability of sclerotia and increased C. minitans infection of the sclerotia of S. sclerotiorum by in comparison with either MP or conidial suspension treatments applied at lower rates (10³-10⁴ cfu cm⁻³ soil). Additionally, when applied at the same rate, MP inoculum of C. minitans was consistently more effective at reducing carpogenic germination than a conidial suspension. The effect of MP and ground MP at full rate on carpogenic germination was expressed relatively early as those sclerotia recovered before apothecia appeared on the soil surface already had reduced numbers of apothecial initials. In general, there were few differences between the isolates of C. minitans applied as conidial suspensions. Box bioassays carried out at different times of the year indicated that temperature and soil moisture influenced both apothecial production and mycoparasitism. Inoculum concentration of C. minitans and time of application appear to be important factors in reducting apothecial production by S. sclerotiorum.     

Elevated CO2 concentration and nitrogen fertilisation effects on N2O and CH4 fluxes and biomass production of Phleum pratense on farmed peat soil

The effects of elevated CO₂ supply on N₂O and CH₄ fluxes and biomass production of Phleum pratense were studied in a greenhouse experiment. Three sets of 12 farmed peat soil mesocosms (10 cm dia, 47 cm long) sown with P. pratense and equally distributed in four thermo-controlled greenhouses were fertilised with a commercial fertiliser in order to add 2, 6 or 10 g N m⁻². In two of the greenhouses, CO₂ concentration was kept at atmospheric concentration (360 μmol mol⁻¹) and in the other two at doubled concentration (720 μmol mol⁻¹). Soil temperature was kept at 15 °C and air temperature at 20 °C. Natural lighting was supported by artificial light and deionized water was used to regulate soil moisture. Forage was harvested and the plants fertilised three times during the basic experiment, followed by an extra fertilisations and harvests. At the end of the experiment CH₄ production and CH₄ oxidation potentials were determined; roots were collected and the biomass was determined. From the three first harvests the amount of total N in the aboveground biomass was determined. N₂O and CH₄ exchange was monitored using a closed chamber technique and a gas chromatograph. The highest N₂O fluxes (on average, 255 μg N₂O m⁻² h⁻¹ during period IV) occurred just after fertilisation at high water contents, and especially at the beginning of the growing season (on average, 490 μg N₂O m⁻² h⁻¹ during period I) when the competition of vegetation for N was low. CH₄ fluxes were negligible throughout the experiment, and for all treatments the production and oxidation potentials of CH₄ were inconsequential. Especially at the highest rates of fertilisation, the elevated supply of CO₂ increased above- and below-ground biomass production, but both at the highest and lowest rates of fertilisation, decreased the total amount of N in the aboveground dry biomass. N₂O fluxes tended to be higher under doubled CO₂ concentrations, indicating that increasing atmospheric CO₂ concentration may affect N and C dynamics in farmed peat soil.     

Transgenic Bt plants decompose less in soil than non-Bt plants

Bt plants are plants that have been genetically modified to express the insecticidal proteins (e.g. Cry1Ab, Cry1Ac, Cry3A) from subspecies of the bacterium, Bacillus thuringiensis (Bt), to kill lepidopteran pests that feed on corn, rice, tobacco, canola, and cotton and coleopteran pests that feed on potato. The biomass of these transgenic Bt plants (Bt+) was decomposed less in soil than the biomass of their near-isogenic non-Bt plant counterparts (Bt−). Soil was amended with 0.5, 1, or 2% (wt wt⁻¹) ground, dried (50 °C) leaves or stems of Bt corn plants; with 0.5% (wt wt⁻¹) ground, dried biomass of Bt rice, tobacco, canola, cotton, and potato plants; with biomass of the near-isogenic plants without the respective cry genes; or not amended. The gross metabolic activity of the soil was determined by CO₂ evolution. The amounts of C evolved as CO₂ were significantly lower from soil microcosms amended with biomass of Bt plants than of non-Bt plants. This difference occurred with stems and leaves from two hybrids of Bt corn, one of which had a higher C:N ratio than its near-isogenic non-Bt counterpart and the other which had essentially the same C:N ratio, even when glucose, nitrogen (NH₄NO₃), or glucose plus nitrogen were added with the biomass. The C:N ratios of the other Bt plants (including two other hybrids of Bt corn) and their near-isogenic non-Bt counterparts were also not related to their relative biodegradation. Bt corn had a significantly higher lignin content than near-isogenic non-Bt corn. However, the lignin content of the other Bt plants, which was significantly lower than that of both Bt and non-Bt corn, was generally not statistically significantly different, although 10-66% higher, from that of their respective non-Bt near-isolines. The numbers of culturable bacteria and fungi and the activity of representative enzymes involved in the degradation of plant biomass were not significantly different between soil amended with biomass of Bt or non-Bt corn. The degradation of the biomass of all Bt plants in the absence of soil but inoculated with a microbial suspension from the same soil was also significantly less than that of their respective inoculated non-Bt plants. The addition of streptomycin, cycloheximide, or both to the soil suspension did not alter the relative degradation of Bt+ and Bt− biomass, suggesting that differences in the soil microbiota were not responsible for the differential decomposition of Bt+ and Bt− biomass. All samples of soil amended with biomass of Bt plants were immunologically positive for the respective Cry proteins and toxic to the larvae of the tobacco hornworm (Manduca sexta), which was used as a representative lepidopteran in insect bioassays (no insecticidal assay was done for the Cry3A protein from potato). The ecological and environmental relevance of these findings is not clear.     

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.     

Growth promoting effects of corn (Zea mays) bacterial isolates under greenhouse and field conditions

Fertilizer costs are a major component of corn production. The use of biofertilizers may be one way of reducing production costs. In this study we present isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3). All bacterial strains produced IAA in the presence of 100 mg l⁻¹ of tryptophan and antifungal metabolites to several soilborne pathogens. S. maltophilia and E. cloacae had broad spectrum activity against most Fusarium species. The only strain that was positive for nitrogen fixation was E. cloacae and it, and P. putida, were also positive for phosphate solubilization. These bacteria and the corn isolate Sphingobacterium canadense CR11, and known plant growth promoting bacterium Burkholderia phytofirmans E24 were used to inoculate corn seed to examine growth promotion of two lines of corn, varieties 39D82 and 39M27 under greenhouse conditions. When grown in sterilized sand varieties 39M27 and 39D82 showed significant increases in total dry weights of root and shoot of 10-20% and 13-28% and 17-32% and 21-31% respectively. Plants of the two varieties grown in soil collected from a corn field had respective increases in dry weights of root and shoot of 10-30% and 12-35% and 11-19% and 10-18%. In sand, a bacterial mixture was highly effective whereas in soil individual bacteria namely P. putida CR7 and E. cloacae CR1 gave the best results with 39M27 and 39D82 respectively. These isolates and another corn isolate, Azospirillum zeae N7, were tested in a sandy soil with a 55 and 110 kg ha⁻¹ of nitrogen fertility at the Delhi research Station of Agriculture and Agri-Food Canada over two years. Although out of seven bacterial treatments, no treatment provided a statistically significant yield increase over control plots but S. canadense CR11 and A. zeae N7 provided statistically significant yield increase as compared to other bacteria. The 110 kg rate of nitrogen provided significant yield increase compared to the 55 kg rate in both years.     

Suppression of damping-off of cucumber caused by Pythium ultimum with live cells and extracts of Serratia marcescens N4-5

Environmentally friendly control measures are needed for the soil-borne pathogen, Pythium ultimum. This pathogen can cause severe losses to field- and greenhouse-grown cucumber and other cucurbits. Live cells and ethanol extracts of cultures of the bacterium Serratia marcescens N4-5 provided significant suppression of damping-off of cucumber caused by P. ultimum when applied as a seed treatment. Live cells of this bacterium also suppressed damping-off caused by P. ultimum on cantaloupe, muskmelon, and pumpkin. Culture filtrates from strain N4-5 contained chitinase and protease activities while ethanol extracts contained the antibiotic prodigiosin, the surfactant serrawettin W1, and possibly other unidentified surfactants. Production of prodigiosin and serrawettin W1 was temperature-dependent, both compounds being detected in extracts from N4-5 grown at 28 °C but not in extracts from N4-5 grown at 37 °C. Ethanol extracts from strain N4-5 grown at 28 °C inhibited germination of sporangia and mycelial growth by P. ultimum in in vitro experiments. There was no in vitro inhibition of P. ultimum associated with ethanol extracts of strain N4-5 grown at 37 °C. Prodigiosin, purified from two consecutive thin-layer chromatography runs using different solvent systems, inhibited germination of sporangia and mycelial growth of P. ultimum. Another unidentified compound(s) also inhibited germination of sporangia but did not inhibit mycelial growth. There was no in vitro inhibition associated with serrawettin W1. These results demonstrate that live cells and cell-free extracts of S. marcescens N4-5 are effective for suppression of damping-off of cucumber caused by P. ultimum possibly due in part to the production of the antibiotic prodigiosin.     

Temperature sensitivity of respiration differs among forest floor layers in a Pinus resinosa plantation

Decomposer microorganisms contribute to carbon loss from the forest floor as they metabolize organic substances and respire CO₂. In temperate and boreal forest ecosystems, the temperature of the forest floor can fluctuate significantly on a day-to-night or day-to-day basis. In order to estimate total respiratory CO₂ loss over even relatively short durations, therefore, we need to know the temperature sensitivity (Q₁₀) of microbial respiration. Temperature sensitivity has been calculated for microbes in different soil horizons, soil fractions, and at different depths, but we would suggest that for some forests, other ecologically relative soil portions should be considered to accurately predict the contribution of soil to respiration under warming. The floor of many forests is heterogeneous, consisting of an organic horizon comprising a few more-or-less distinct layers varying in decomposition status. We therefore determined at various measurement temperatures the respiration rates of litter, F-layer, and H-layer collected from a Pinus resinosa plantation, and calculated Q₁₀ values for each layer. Q₁₀ depended on measurement temperature, and was significantly greater in H-layer than in litter or F-layer between 5 and 17 °C. Our results indicate, therefore, that as the temperature of the forest floor rises, the increase in respiration by the H-layer will be disproportionate to the increase by other layers. However, change in respiration by the H-layer associated with change in temperature may contribute minimally or significantly to changes of total forest floor respiration in response to changes in temperature depending on the depth and thickness of the layer in different forest ecosystems.     

Kinetics of bacterial death by heated dolomite powder slurry

The kinetics of the bactericidal action of dolomite powders heated at 600-1000 °C against Escherichia coli and Staphylococcus aureus were investigated. Dolomite powder heated to at least 700 °C exhibited bactericidal action, and the process of bacterial death in the heated dolomite powder slurries followed first-order reaction kinetics. The value of the death rate constant (k) increased with dolomite powder concentration, and the dilution coefficient (n), which indicates the dependence of k on the reagent concentration, was measured. The n values of the powder heated at 700 °C and at temperatures >900 °C were almost identical to those of MgO and CaO, respectively. This suggests that the first emergence of bactericidal action at 700 °C corresponds to generation of MgO while that at temperatures >900 °C is due to generation of CaO. The slurry temperature significantly affects the bactericidal action. The slope of the Arrhenius plot of k for E. coli and S. aureus grown at 37 °C exhibited a discontinuous point at approximately 22 °C, where a change in the value of activation energy for bacterial death occurred. This temperature corresponds to that of the phase transition of cell membrane lipids.     

Nitrous oxide emissions from agricultural soils at low temperatures: a laboratory microcosm study

We studied in laboratory microcosms (intact soil cores) N₂O and CO₂ emissions from four different agricultural soil types (organic soil, clay, silt and loam) at low temperatures with or without freezing-thawing events. When the temperature of the frozen soil cores was increased stepwise from −8 °C the N₂O emissions began to increase at −0.5 °C, and peaked at −0.1 °C in the organic, clay and silt soils, and at +1.6 °C in the loam soils. However, a stepwise decrease in soil temperature from +15 °C also induced an increase in the N₂O emissions close to the 0 °C. These emissions peaked between −0.4 and +2.5 °C depending on the soil type and water content. However, the emission maxima were from 2 to 14.3% of those encountered in the experiments where frozen soils were thawed. Our results show that in addition to the well-documented thawing peak, soils also can have a maximum in their N₂O emission near 0 °C when soil temperature decrease. These emissions, however, are less than those emitted from thawing soils. The correlations between the N₂O and CO₂ emissions were weak. Our results suggest that N₂O is produced in soils down to a temperature of −6 °C.     

Unfrozen water content moderates temperature dependence of sub-zero microbial respiration

Abrupt increases in the temperature sensitivity of soil respiration below 0 °C have been interpreted as a change in the dominance of other co-dependent environmental controls, such as the availability of liquid-state water. Yet the relationship between unfrozen water content and soil respiration at sub-zero temperatures has received little attention because of difficulties in measuring unfrozen water contents. Using a recently-developed semi-solid ²H NMR technique the unfrozen water content present in seasonally frozen boreal forest soils was quantified and related to biotic CO₂ efflux in laboratory microcosms maintained at temperatures between −0.5 and −8 °C. In both soils the unfrozen water content had an exponential relationship with temperature and was increased by addition of KCl solutions of defined osmotic potential. Approximately 13% unfrozen water was required to release the dependence of soil respiration on unfrozen water content. Depending on the osmotic potential of soil solution, this threshold unfrozen water content was associated with temperatures down to −6 °C; yet if temperature were the predictor of CO₂ efflux, then the abrupt increase in the temperature sensitivity of CO₂ efflux was associated with −2 °C, except in soils amended with −1500 kPa KCl which did not show any abrupt changes in temperature sensitivity. The KCl-amendments also had the effect of decreasing Q₁₀ values and activation energies (Ea) by factors of 100 and three, respectively, to values comparable with those for soil respiration in unfrozen soil. The disparity between the threshold temperatures and the reductions in Q₁₀ values and activation energies after KCl amendment indicates the significance of unfrozen water availability as an environmental control of equal importance to temperature acting on sub-zero soil respiration. However, this significance was diminished when soils were supplied with abundant labile C (sucrose) and the influences of other environmental controls, allied to the solubility and diffusion of respiratory substrates and gases, are considered to increase.     

Time and burial depth influencing the viability and bacterial colonization of sclerotia of Sclerotinia sclerotiorum

Sclerotia are the primary over wintering inoculum of Sclerotinia sclerotiorum (Lib.) de Bary. The effects of tillage on the primary inoculum are not well understood. The purpose of this research was to study sclerotial viability over time and between burial depths in soil, to identify bacteria colonizing and degrading the sclerotia, and determine whether these bacteria may be utilized as biological control agents. Correlation analysis indicated that a significant negative relationship existed between sclerotial viability and elapsed temporal factors (R²=−0.68, P<0.0001), and depth of burial (R²=−0.58, P<0.0001). After twelve months, sclerotia on the soil surface had the highest viability (57.5%), followed by those at the 5 cm depth (12.5%), and only 2.5% of those placed at the 10 cm depth remained viable. A significant negative relationship between sclerotial viability and bacterial populations also existed (R²=−0.60, P<0.0001). Two hundred and sixty-eight bacteria were isolated from sclerotia, 29 of which showed strong in vitro antagonism to the mycelial growth of S. sclerotiorum. Biodiversity of the inhibitory bacterial isolates was minimal on sclerotia from the soil surface and within all depths sampled at three months (i.e. in January). All burial depths within the April and July sampling dates produced bacterial diversities that were distinct from each other.     

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).     

Changes in mineral nitrogen, phosphorus availability and salt-extractable aluminium following the application of green manure residues in two weathered soils of South Vietnam

Phosphorus deficiency and aluminium toxicity in weathered soils can be amended by applying organic residues. Nitrogen mineralization, changes in P-availability and changes in salt-extractable Al following the incorporation of residues of various green manures (Flemingia congesta, Mucuna pruriens, Pueraria phaseoloides, Tithonia diversifolia) were quantified in a field core incubation experiment. Dried residues were added at an application rate of 45 kg P ha⁻¹ to two soils representative for the main soil groups of the South Vietnamese uplands, set up in incubation cores in an experimental field near Ho Chi Minh City, Vietnam.Decomposition of the residues proceeded at high rates. Mineralized nitrogen from the residues was recovered mainly as ammonium during the first 2 weeks of incubation. Nitrogen release from Tithonia residues with the highest lignin content and lignin:N ratio occurred more gradually compared to the three legumes. Resin-extractable P was significantly increased by residue treatments. Largest and sustained increases in resin-extractable P (0.67 and 2.06 mg P kg⁻¹ in the two soils) were observed in samples amended with Tithonia, which was related to the large P-content (0.37%) and small C:P ratio (110) of the residues. The P-concentration in the residues, rather than the total amount of P applied through the residues, affected the increase in P-availability. The increase in resin-extractable P was correlated to the P-content (R=0.64) and C:P ratio (R=−0.65) of the residues. Salt-extractable Al-concentrations were considerably reduced by the organic amendments, up to 70 and 50% in the two soils. At the rate of 45 kg P ha⁻¹, no significant differences between the residue treatments to reduce soil acidity were observed.As such, the application of high quality residues that are rich in P, in particular T. diversifolia, may enhance crop production by creating favourable soil conditions during the initial stages of plant development of the main crop.     

Interaction between the soil yeast Rhodotorula mucilaginosa and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.     

Feeding activity of the earthworm Eisenia andrei in artificial soil

Quantitative information on the feeding activity of earthworms is scarce but this information is valuable in many eco(toxico)logical studies. In this study, the feeding activity of the compost worm Eisenia andrei is examined in artificial soil (OECD medium), with and without a high-quality food source (cow manure), and at two temperatures (10 and 20 °C). Methods are provided to estimate the most important parameters: gut load, selection of organic matter (OM), digestion efficiency, compaction, gut retention time, and fraction of manure in the diet. Lanthanides (Lu and Tm) were successfully used as inert markers in soil and manure, and we applied Bayesian statistics to analyse the data and fully capture the compounded uncertainty in the parameter estimates. Results show that the compost worm does not feed on soil indiscriminately but is able to select an OM-enriched diet from apparently homogeneous OECD medium. When manure is present on the soil surface, approximately three-quarters of the diet still consists of soil particles. The gut load of the worms was approximately 10% (dwt gut/wwt empty worm), varying little with the treatments. Unfortunately, the digestion efficiency could only be reliably estimated at 20 °C, and was approximately 40%. Temperature clearly affected feeding as a 10° temperature decrease nearly doubled the gut retention time (from 2.9 to 5.5 h), which corresponds to a two-fold decrease in feeding rate. The present data may be used to interpret toxicity and accumulation studies with E. andrei in OECD medium. However, care must be taken, as it seems possible that feeding is influenced by the size of the worm and subtle differences in experimental set-up.     

Soil texture and irrigation influence the transport and the development of Pasteuria penetrans, a bacterial parasite of root-knot nematodes

The transport of the spores of Pasteuria penetrans was studied in three contrasted textured soils (a sandy, a sandy-clay and a clay soils), cultivated with tomato, inoculated with juveniles of Meloidogyne javanica and watered with 25 or 150 mm day⁻¹. One month after inoculation of the nematodes, 53% of the spores inoculated were leached by water flow in the sandy soil but only 14% in the sandy-clay soil and 0.1% in the clay soil. No nematodes survived in the clay soil, while the population was multiplied both in the sandy and in the sandy-clay soils. But juveniles of M. javanica were more infected by P. penetrans in the sandy-clay soil than in the sandy soil. Comparing different combinations of bare soils containing 1.1-57% of clay showed that the best spore percolation and retention balance occurred in soils amended with 10-30% clay. However, the spore recoveries decreased when the soil was enriched with more than 30% clay. The role of clay particles on the extractability of spores and on their availability to attach to the nematode cuticle in the soil is discussed.     

Thermal stability of soil organic matter pools and their δC values after C3-C4 vegetation change

Carbon isotopic composition of soils subjected to C₃-C₄ vegetation change is a suitable tool for the estimation of C turnover in soil organic matter (SOM) pools. We hypothesized that the biological availability of SOM pools is inversely proportional to their thermal stability. Soil samples from a field plot with 10.5 years of cultivation of the C₄ plant Miscanthus×gigantheus and from a reference plot under C₃ grassland vegetation were analysed by thermogravimetry coupled with differential scanning calorimetry (TG-DSC). According to differential weight losses (dTG) and energy release or consumption (DSC), five SOM pools with increasing thermal stability were distinguished: (I) 20-190 °C, (II) 190-310 °C, (III) 310-390 °C, (IV) 390-480 °C, and (V) 480-1000 °C. Their δ¹³C values were analysed by EA-IRMS. The weight losses in pool I were connected with water evaporation, since no significant C losses were measured and δ¹³C values remained unchanged. The δ¹³C of pools II and III in soil samples under Miscanthus were closer to the δ¹³C of the Miscanthus plant tissues (−11.8‰) compared to the thermally stable SOM pool V (−19.5‰). The portion of the Miscanthus-derived C₄-C in total SOM in 0-5 cm reached 55.4% in the 10.5 years. The C₄-C contribution in pool II was 60% and decreased down to 6% in pool V. The mean residence times (MRT) of SOM pools II, III, and IV were similar (11.6, 12.2, and 15.4 years, respectively), while pool V had a MRT of 163 years. Therefore, we concluded that the biological availability of thermal labile SOM pools (<480 °C) was higher, than that of the thermal stable pool decomposed above 480 °C. However, the increase of SOM stability with rising temperature was not gradual. Therefore, the applicability of the TG-DSC for the separation of SOM pools with different biological availability is limited.