Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum

The filamentous fungi Trichoderma spp. is currently developed as biocontrol agents against many plant pathogens. Recent studies have shown that these fungi are able to infect nematode eggs and juveniles. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Trichoderma harzianum BI was investigated in greenhouse and laboratory experiments. Results showed that different concentrations (10²–10⁸ spores/ml) of T. harzianum BI decreased nematode infection and other parameters significantly, compared to control. T. harzianum BI was able to penetrate nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased significantly in T. harzianum BI inoculated plants. Maximum activities of POX, PPO and PAL were observed at the 5, 5 and 6 days after inoculation, respectively. Chitinase activity was also increased in culture filtrates of T. harzianum BI grown on wheat bran moistened with salt solution supplemented with colloidal chitin or nematode eggs. Maximum activity of chitinase was recorded at the 4 days after inoculation, in media supplemented with colloidal chitin (1.15 U/min per ml) and nematode eggs (0.85 U/min per ml). Results suggested that direct parasitism of eggs through the increase in extracellular chitinase activity, which would be indicator of eggs infection capability, and inducing plant defense mechanisms leading to systemic resistance are two main suppression mechanisms used by T. harzianum BI against nematode.     

Effect of sclerotial distribution pattern of Sclerotinia sclerotiorum on biocontrol efficacy of Trichoderma harzianum

Field studies were conducted over two seasons to investigate effects of random versus highly aggregated spatial arrangements of sclerotia of S. sclerotiorum, and effects of biocontrol agent density and formulation additives, on colonization of sclerotia by Trichoderma spp. Application of T. harzianum encapsulated in alginate pellets with either bran or polyethylene glycol additives increased the percentages of sclerotia colonized in both years, but there was no difference between additives in either year. Higher pellet densities (200 pellets/m² versus 40 pellets/m²) resulted in higher proportions of sclerotia colonized by Trichoderma spp. in one season but not in the other. However, when sclerotia were in highly aggregated spatial patterns, significantly higher percentages were colonized in both years, compared to sclerotia in random distributions.     

Removal of benzo (a) pyrene from soil using an endogeic earthworm Pontoscolex corethrurus ()

The endogeic earthworm Pontoscolex corethrurus (Müller, 1857) was the most abundant species (75%) in soil contaminated with hydrocarbons, mostly benzo(a)pyrene (BaP), in the state of Tabasco (Mexico). The earthworm P. corethrurus was tested for its capacity to remove 100 mg BaP kg⁻¹ from an Anthrosol soil (sterilized or not) and amended with legume Mucuna pruriens (L.) DC. var. utilis (Wall. ex Wight) Baker ex Burck (3%) or the grass Brachiaria humidicola (L.) DC (3%) (recently renamed as Urochloa humidicola (Rendle) Morrone & Zuloaga) in an aerobic incubation experiment. P. corethrurus removed 26.6 mg BaP kg⁻¹ from the sterilized soil and application of B. humidicola as feed increased this to 35.7 mg BaP kg⁻¹ and M. pruriens to 34.2 mg BaP kg⁻¹ after 112 days. The autochthonous microorganisms removed 9.1 mg BaP kg⁻¹ from the unsterilized soil and application of B. humidicola increased this to 18.0 mg BaP kg⁻¹ and M. pruriens to 11.2 mg BaP kg⁻¹. Adding P. corethrurus to the unsterilized soil accelerated the removal of BaP and 36.1 mg kg⁻¹ was dissipated from soil. It was found that the autochthonous microorganisms removed BaP from soil, but addition of P. corethrurus increased the dissipation 4-fold. The endogeic earthworm P. corethrurus can thus be used to remediate hydrocarbon-contaminated soils in tropical regions.     

Effects of two soil reclamation techniques on the distribution of the organic N compounds in a 15N labelled burnt soil

The evolution of the soil organic-N forms and their bio-availability was studied in a ¹⁵N labelled and burnt soil (BLS) after two successive reclamation steps under greenhouse conditions: a 3-month growing period of Lolium, without (BLS-L) or with poultry manure addition (4 and 8 Mg ha^− 1: BLS + PM4-L and BLS + PM8-L), followed by a 12-month growing phase of pine seedlings (BLS-P, BLS + PM4-P and BLS + PM8-P). The results were compared with those obtained for the homologous labelled unburnt soil (LS, LS-L and LS-P) to evaluate the efficacy of these reclamation techniques in the mitigation of the drastic post-fire changes exhibited by the major biologically available N pool in terrestrial ecosystems: the soil organic N. The significant and steady decrease of the ¹⁵N enrichment observed in the unburnt soil during the successive plant growth cycles (LS > LS-L > LS-P) contrasts with the lack of significant changes, in both the content of total organic ¹⁵N and the atom % ¹⁵N in excess, among the treatments with the burnt soil (BLS ≥ BLS-L ≥ BLS-P). These results showed that: a) in LS, N mineralization proceeds faster for the recently incorporated N (¹⁵N enriched) than for the native N, supplying the growing vegetation with inorganic N more ¹⁵N enriched than the bulk soil N; and b) in BLS, soil combustion has reduced the usually higher biological availability of the recently added N to levels similar to those of the endogenous N.The re-vegetation with Lolium and Pinus and the addition of poultry manure mitigated the high differences observed in the size of the amino acid and the organic derived NH₄⁺–N pools due to the combustion process, which are usual between burnt and unburnt soils. Conversely, these burnt soil reclamation techniques (re-vegetation and poultry manure addition), even jointly used, were unable to reduce the huge differences observed between the burnt and the unburnt soils for the other N fractions considered (amides, amino sugars, hydrolysable unidentified-N, hydrolysable organic N and un-hydrolysable N) that accounted for more than 80% of the soil organic N. Consequently, it seems that without the introduction of N₂-fixing microorganisms or plants in the burnt soils the recovery of the natural soil organic N composition will take place slowly.     

Contribution of plant photosynthate to soil respiration and dissolved organic carbon in a naturally recolonising cutover peatland

The aim of this study was to investigate how three vascular plant species (Calluna vulgaris, Eriophorum angustifolium and Eriophorum vaginatum) colonising an abandoned cutover peatland affect fluxes of recent photosynthate to dissolved organic carbon (DOC), soil and plant respiration and shoot biomass. We used in situ ¹³CO₂ pulse labelling to trace carbon (C) throughout a 65 day pulse chase period. Between 16 and 35% of the pulse of ¹³C remained in shoot biomass after 65 days with significant differences between C. vulgaris and E. angustifolium (P = 0.009) and between C. vulgaris and E. vaginatum (P = 0.04). A maximum of 29% was detected in DOC beneath labelled plants and losses of ¹³C from peat respiration never exceeded 0.16% of the original pulse, showing that little newly fixed C was allocated to this pool. There were no significant differences between the different plant species with respect to ¹³C recovered from DOC or via peat respiration. More C was lost via shoot respiration; although amounts varied between the three plant species, with 4.94–27.33% of the ¹³C pulse respired by the end of the experiment. Significant differences in ¹³C recovered from shoot respiration were found between C. vulgaris and E. angustifolium (P = 0.001) and between E. angustifolium and E. vaginatum (P = 0.032). Analysis of δ¹³C of microbial biomass indicated that recently assimilated C was allocated to this pool within 1 day of pulse labelling but there were no significant differences in the ¹³C enrichment of the microbial biomass associated with the different plant species. The data suggest that peat respiration represents a small flux of recent assimilate compared to other fluxes and pools and that different vascular plant species show considerable variation in the quantities and dynamics of C allocated to DOC.     

Fungal communities in fallow soil before and after amending with pine sawdust

Pine sawdust (9 kg m⁻²) was ploughed into soils, intended for future forestry plantations, that had been left for 3 or 6 years after previously being used for agriculture. Two years after the amendment, soil pH had changed in both sites and the C:N ratio had increased. Total fungal populations decreased after the treatment. A small decrease in the number of species was not statistically significant. Trichoderma harzianum, an important biological control fungus, increased considerably after sawdust application whilst some fungi, such as Penicillium spp., Pseudogymnascus roseus and partially Mucorales decreased. The practical implications of the increase in T. harzianum density for the health of the young forests are discussed.     

The role of biological activity (roots,earthworms) in medium-term C dynamics in vertisol under a Digitaria decumbens (Gramineae) pasture

The natural abundance of ¹³C was used to estimate the turnover of the soil organic matter in a vertisol re-grassed with Digitaria decumbens (C4 plant) following intensive market gardening (C3 plants). In addition, the experimental design allowed us to determine the respective roles of roots and earthworms (Polypheretima elongata) in soil C stock restoration in D. decumbens pasture.The C stock increased from 31 to 37 Mg C ha⁻¹ in 5 years and the δ¹³C increased from −18.1‰ in market gardening soil to −15.5‰ in the 5-year-old pasture soil in the upper 20 cm. Below the 20 cm soil layer, the C stock and the δ¹³C did not change significantly in 5 years. The net gain of 6 Mg C ha⁻¹ was the balance of a loss of 5 Mg C ha⁻¹ derived from market gardening and a gain of 11 Mg C ha⁻¹ derived from D. decumbens. Effects of earthworms on the C dynamics were not discernible.     

The rhizosphere soil of diseased tomato plants as a source for novel microorganisms to control bacterial wilt

Plant growth promoting rhizobacteria (PGPRs) are used for biocontrol of bacterial wilt caused by Ralstonia solanacearum. They are commonly isolated from the rhizosphere of healthy plants and are scarce in the rhizosphere of diseased plants. We hypothesized that a pathogen-prevalent environment, such as the rhizosphere of infected plants, would be a good or better source for isolating PGPRs than the rhizosphere of healthy plants. In order for these PGPRs to survive successfully in a pathogen-prevalent environment, they must have particularly well-developed survival strategies under the stresses exerted by pathogen activities, which would be of value for their use as biocontrol agents. To test this hypothesis, R. solanacearum-antagonistic bacteria were screened from the rhizospheres of diseased and healthy tomato plants. In total, 110 rhizobacteria were isolated, 18 of which showed antagonism to R. solanacearum in vitro. Among the 18 antagonistic strains, 11 (out of 60) were from the rhizosphere of diseased plants, with inhibition diameter zones ranging from 11.2 to 15.2 mm, whereas 7 (out of 50) were from the rhizosphere of healthy plants, with inhibition diameter zones ranging from 11.5 to 30.5 mm. Strains WR4, WR21, and WR42 from diseased plants rhizosphere, and HR61, HR62, and HR92 from healthy plants rhizosphere, were chosen to investigate their biocontrol efficacies (BCEs) in greenhouse condition. Results showed that WR-isolates performed better in reducing disease incidence (DI) than those HR-isolates. Population densities of R. solanacearum in the rhizosphere soil and crown section of tomato plants were lower in WR-isolate treatments than those in HR-isolate treatments. The best biocontrol effect was achieved by inoculating the strain WR21, followed by WR4, WR42, HR92, HR62, and HR61. Root colonization test showed WR21 had the highest root-colonizing capacity compared with 5 other antagonists. BCEs were positively (r = 0.747) correlated with root-colonizing capacities, but were negatively (r = −0.797) correlated with inhibition zones. In conclusion, the rhizosphere of diseased tomato plants is a good reservoir of biocontrol bacteria.     

Soil organic matter in soil depth profiles: Distinct carbon preferences of microbial groups during carbon transformation

This study investigates how carbon sources of soil microbial communities vary with soil depth. Microbial phospholipid fatty acids (PLFA) were extracted from 0–20, 20–40 and 40–60 cm depth intervals from agricultural soils and analysed for their stable carbon isotopes (δ¹³C values). The soils had been subjected to a vegetation change from C3 (δ¹³C≈?29.3‰) to C4 plants (δ¹³C≈?12.5‰) 40 years previously, which allowed us to trace the carbon flow from plant-derived input (litter, roots, and root exudates) into microbial PLFA. While bulk soil organic matter (SOM) reflected ≈12% of the C4-derived carbon in top soil (0–20 cm) and 3% in deeper soil (40–60 cm), the PLFA had a much higher contribution of C4 carbon of about 64% in 0–20 cm and 34% in 40–60 cm. This implies a much faster turnover time of carbon in the microbial biomass compared to bulk SOM. The isotopic signature of bulk SOM and PLFA from C4 cultivated soil decreases with increasing soil depth (?23.7‰ to ?25.0‰ for bulk SOM and ?18.3‰ to ?23.3‰ for PLFA), which demonstrates decreasing influence of the isotopic signature of the new C4 vegetation with soil depth. In terms of soil microbial carbon sources this clearly shows a high percentage of C4 labelled and thus young plant carbon as microbial carbon source in topsoils. With increasing soil depth this percentage decreases and SOM is increasingly used as microbial carbon source. Among all PLFA that were associated to different microbial groups it could be observed that (a) depended on availability, Gram-negative and Gram-positive bacteria prefer plant-derived carbon as carbon source, however, (b) Gram-positive bacteria use more SOM-derived carbon sources while Gram-negative bacteria use more plant biomass. This tendency was observed in all three-depth intervals. However, our results also show that microorganisms maintain their preferred carbon sources independent on soil depth with an isotopic shift of 3–4‰ from 0–20 to 40–60 cm soil depth.     

Partitioning soil surface CO2 efflux into autotrophic and heterotrophic components,using natural gradients in soil δ13C in an undisturbed savannah soil

We used natural gradients in soil and vegetation δ¹³C signatures in a savannah ecosystem in Texas to partition soil respiration into the autotrophic (Ra) and heterotrophic (Rh) components. We measured soil respiration along short transects from under clusters of C₃ trees into the C₄ dominated grassland. The site chosen for the study was experiencing a prolonged drought, so an irrigation treatment was applied at two positions of each transect. Soil surface CO₂ efflux was measured along transects and CO₂ collected for analysis of the δ¹³C signature in order to: (i) determine how soil respiration rates varied along transects and were affected by localised change in soil moisture and (ii) partition the soil surface CO₂ efflux into Ra and Rh, which required measurement of the δ¹³C signature of root- and soil-derived CO₂ for use in a mass balance model.The soil at the site was unusually dry, with mean volumetric soil water content of 8.2%. Soil respiration rates were fastest in the centre of the tree cluster (1.5 ± 0.18 μmol m^?2 s^?1; mean ± SE) and slowest at the cluster–grassland transition (0.6 ± 0.12 μmol m^?2 s^?1). Irrigation produced a 7–11 fold increase in the soil respiration rate. There were no significant differences (p > 0.5) between the δ¹³C signature of root biomass and respired CO₂, but differences (p < 0.01) were observed between the respired CO₂ and soil when sampled at the edge of the clusters and in the grassland. Therefore, end member values were measured by root and soil incubations, with times kept constant at 30 min for roots and 2 h for soils. The δ¹³C signature of the soil surface CO₂ efflux and the two end member values were used to calculate that, in the irrigated soils, Rh comprised 51 ± 13.5% of the soil surface CO₂ efflux at the mid canopy position and 57 ± 7.4% at the drip line. In non-irrigated soil it was not possible to partition soil respiration, because the δ¹³C signature of the soil surface CO₂ efflux was enriched compared to both the end member values. This was probably due to a combination of the very dry porous soils at our study site (which may have been particularly susceptible to ingress of atmospheric CO₂) and the very slow respiration rates of the non-irrigated soils.     

An outline of soil nematode succession on abandoned fields in South Bohemia

Secondary succession of nematodes was studied in 1–48-year-old abandoned fields on cambisols in South Bohemia, Czech Republic, and compared with cultivated field and sub-climax oak forests. Bacterivores were the predominant group in the cultivated field whereas in forests root-fungal feeders (mainly Filenchus) were almost as abundant as bacterivores. The total abundance of nematodes in the cultivated field averaged 868 × 10³ ind m⁻². During the first three years of succession the abundance practically did not change (775 × 10³ ind m⁻²), the fauna was still similar to that in cultivated field but the biomass increased mainly due to Aporcelaimellus. Then the abundance increased up to 3731 × 10³ ind m⁻² in 7–8-year-old abandoned fields, plant parasites (Helicotylenchus) dominated and the fungal-based decomposition channel was activated. Later the abundance stabilised at between 1086 and 1478 × 10³ ind m⁻² in 13–25-year-old successional meadow stages with high population densities of omnivores and predators. The total abundance of nematodes was low in the 12–13-year-old willow shrub stage (594 × 10³ ind m⁻²), increased in the 35–48-year-old birch shrub stage (1760 × 10³ ind m⁻²) and the nematode fauna developed towards a forest community. The diversity and maturity of nematode communities generally increased with the age of abandoned fields but the highest values were in meadow stages (81–113 species, 57–68 genera, MI 2.73–3.30). The development of meadow arrested succession towards forests or diverted succession towards a waterlogged ecosystem. The succession of nematodes was influenced by the method of field abandonment (bare soil vs. legume cover, mowing) that affected the formation of either a shrub or meadow stage, and by the soil water status. The composition of the nematode fauna indicated that the soil food web could recover faster from agricultural disturbance under successive meadows than under shrubs.     

Mechanisms of short-term soil carbon storage in experimental grasslands

We investigated the fate of root and litter derived carbon in soil organic matter and dissolved organic matter in soil profiles, in order to explain mechanisms of short-term soil carbon storage. A time series of soil and soil solution samples was investigated at the field site of The Jena Experiment between 2002 and 2004. In addition to the main experiment with C3 plants, a C4 species (Amaranthus retroflexus L.) naturally labeled with ¹³C was grown on an extra plot. Changes in organic carbon concentration in soil and soil solution were combined with stable isotope measurements to follow the fate of plant carbon into the soil and soil solution. A split plot design with plant litter removal versus double litter input simulated differences in biomass input. After 2 years, the no litter and double litter treatment, respectively, showed an increase of 381 g C m^?2 and 263 g C m^?2 to 20 cm depth, while 71 g C m^?2 and 393 g C m^?2 were lost between 20 and 30 cm depth. The isotopic label in the top 5 cm indicated that 115 g C m^?2 and 156 g C m^?2 of soil organic carbon were derived from C4 plant material on the no litter and the double litter treatment, respectively. Without litter, this equals the total amount of 97 g C m^?2 that was newly stored in the same soil depth, whereas with double litter this clearly exceeded the stored amount of 75 g C m^?2. Our results indicate that litter input resulted in lower carbon storage and larger carbon losses and consequently accelerated turnover of soil organic carbon. Isotopic evidence showed that inherited soil organic carbon was replaced by fresh plant carbon near the soil surface. Our results suggest that primarily carbon released from soil organic matter, not newly introduced plant organic matter, was transported in the soil solution. However, the total flow of dissolved organic carbon was not sufficient to explain the observed carbon storage in deeper soil layers, and the existence of additional carbon uptake mechanisms is discussed.     

Plant growth promoting potential of Pontibacter niistensis in cowpea (Vigna unguiculata (L.) Walp.)

During the past couple of decades, understanding of rhizosphere biology has progressed with the discovery of a special group of microorganisms known as plant growth promoting rhizobacteria (PGPR) and its application for sustainable agriculture has increased tremendously in various parts of the world. The search for microorganisms that improve soil fertility and enhance plant nutrition has continued to attract attention due to the increasing cost of fertilizers and some of their negative environmental impacts. In this study we demonstrated, a novel bacterial species Pontibacter niistensis NII-0905 isolated from forest soil in Western ghat forest soil with potential plant growth promoting ability (PGP) such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production. The activity varies with different growth temperatures, strain solubilize 28.5 ± 0.9, 48.02 ± 1.9 and 65.07 ± 2.1 μg mL⁻¹ at 4, 15 and 30 °C respectively and produced 24.8 μg mL⁻¹ day⁻¹ of indole acetic acid (IAA) in tryptophan amended media. Qualitative detection of siderophore production and HCN were also detected at all temperature tested. At a lower temperature (4 °C) strain NII-0905 retained all the plant growth promotion attributes. A significant increase in the growth of cow pea was recorded with inoculations of strain NII-0905 in pot experiments. Scanning electron microscopic study revealed the root colonization on cow pea seedlings against the untreated one. These results demonstrate that, the isolate NII-0905 has the promising PGPR attributes for both in cold as well as in humid condition. It has potential as a biofertilizer to enhance soil fertility and promote the plant growth.     

Resistance to oxidation products of caffeic acid is important for efficient colonization of wheat seedlings by Pseudomonas proteolytica strain PSR114

The interrelationships between plants and rhizosphere bacteria are strongly dependent on the quality and quantity of root exudates. The ability to colonize roots is crucial for pseudomonads to function as biological control agents of root- and soil-borne pathogenic microbes. The multiplication of rhizosphere bacteria is restricted in the presence of simple phenolic compounds, which are components of the resistance mechanisms of plants to pathogens. Caffeic acid is a phenolic compound, which is commonly found in wheat tissues. It is prone to oxidation into o-quinones, which are toxic to microorganisms. The aim of the present study was to determine whether the ability of microorganisms to resist caffeic acid and its oxidation products could play a role in the early colonization of wheat seedlings. Among the fluorescent pseudomonads that we have studied, strain PSR114 is one of the most efficient colonizers of wheat seedlings during the first 48 h after seed germination, and it is particularly resistant to products resulting from the spontaneous oxidation of caffeic acid. This strain was isolated from the rhizosphere of oilseed rape and identified as being closely related to Pseudomonas proteolytica through the analysis of 16S rRNA and rpoB gene sequences. At pH 7.0, this strain grew intensively in the presence of 1.50 mg mL⁻¹ of caffeic acid. Its multiplication was partially reduced in the presence of oxidized caffeic acid at concentrations above 0.21 mg mL⁻¹, and completely inhibited at concentrations above 0.38 mg mL⁻¹. A Tn5 transposon mutant of PSR114 had lower level of resistance to the oxidation products of caffeic acid, as well as reduced capacity to colonize wheat seedlings when compared to the wild type strain. This work demonstrates that resistance to oxidation products of caffeic acid can be important for successful bacterial colonization of wheat seedlings.     

Quantifying potato pathogen DNA in soil

An improved method for the direct extraction of DNA from soil involving processing of a relatively large sample (60 g) was developed. The accurate and reliable detection and quantification of the soil-borne potato pathogens Colletotrichum coccodes (black dot), Rhizoctonia solani (black scurf) and Spongospora subterranea (powdery scab) following inoculation of soils was demonstrated. With this method, low levels of target DNA (30–40 pg DNA/g soil) could be detected in field soils. DNA recovery was proportionate across a wide range of inoculum (R² > 0.86) and there was no effect of soil type on the recovery of C. coccodes. The method was used to assess levels of naturally occurring pathogen DNA in 122 soil samples obtained from commercial potato fields.     

Evapotranspiration and water use efficiency in different-aged Pacific Northwest Douglas-fir stands

We used the eddy-covariance technique to measure evapotranspiration (E) and gross primary production (GPP) in a chronosequence of three coastal Douglas-fir (Pseudotsuga menziesii) stands (7, 19 and 58 years old in 2007, hereafter referred to as HDF00, HDF88 and DF49, respectively) since 1998. Here, we focus on the controls on canopy conductance (g_c), E, GPP and water use efficiency (WUE) and the effect of interannual climate variability at the intermediate-aged stand (DF49) and then analyze the effects of stand age following clearcut harvesting on these characteristics. Daytime dry-foliage Priestley–Taylor α and g_c at DF49 were 0.4–0.8 and 2–6 mm s^?1, respectively, and were linearly correlated (R² = 0.65). Low values of α and g_c at DF49 as well at the other two stands suggested stomatal limitation to transpiration. Monthly E, however, showed strong positive linear correlations to monthly net radiation (R² = 0.94), air temperature (R² = 0.77), and daytime vapour pressure deficit (R² = 0.76). During July–September, monthly E (mm) was linearly correlated to monthly mean soil water content (θ, m³ m^?3) in the 0–60 cm layer (E = 453θ ? 21, R² = 0.69), and GPP was similarly affected. Annual E and GPP of DF49 for the period 1998–2007 varied from 370 to 430 mm and from 1950 to 2390 g C m^?2, respectively. After clearcut harvesting, E dropped to about 70% of that for DF49 while ecosystem evapotranspiration was fully recovered when stand age was ～12 years. This contrasted to GPP, which varied hyperbolically with stand age. Monthly GPP showed a strong positive linear relationship with E irrespective of the stand age. While annual WUE of HDF00 and HDF88 varied with age from 0.5 to 4.1 g C m^?2 kg^?1 and from 2.8 to 4.4 g C m^?2 kg^?1, respectively, it was quite conservative at ～5.3 g C m^?2 kg^?1 for DF49. N-fertilization had little first-year response on E and WUE. This study not only provides important results for a more detailed validation of process-based models but also helps in predicting the influences of climate change and forest management on water vapour and CO₂ fluxes in Douglas-fir forests.     

Comparison of the eddy covariance and automated closed chamber methods for evaluating nocturnal CO2 exchange in a Japanese cypress forest

Underestimation of nocturnal CO₂ respiration using the eddy covariance method under calm conditions remains an unsolved problem at many flux observation sites in forests. To evaluate nocturnal CO₂ exchange in a Japanese cypress forest, we observed CO₂ flux above the canopy (F_c), changes in CO₂ storage in the canopy (S_t) and soil, and trunk and foliar respiration for 2 years (2003–2004). We scaled these chamber data to the soil, trunk, and foliar respiration per unit of ground area (F_s, F_t, F_f, respectively) and used the relationships of F_s, F_t, and F_f with air or soil temperature for comparison with canopy-scale CO₂ exchange measurements (=F_c + S_t). The annual average F_s, F_t, and F_f were 714 g C m⁻² year⁻¹, 170 g C m⁻² year⁻¹, and 575 g C m⁻² year⁻¹, respectively. At small friction velocity (u_*), nocturnal F_c + S_t was smaller than F_s + F_t + F_f estimated using the chamber method, whereas the two values were almost the same at large u_*. We replaced F_c + S_t measured during calm nocturnal periods with a value simulated using a temperature response function derived during well-mixed nocturnal periods. With this correction, the estimated net ecosystem exchange (NEE) from F_c + S_t data ranged from −713 g C m⁻² year⁻¹ to −412 g C m⁻² year⁻¹ in 2003 and from −883 g C m⁻² year⁻¹ to −603 g C m⁻² year⁻¹ in 2004, depending on the u_* threshold. When we replaced all nocturnal F_c + S_t data with F_s + F_t + F_f estimated using the chamber method, NEE was −506 g C m⁻² year⁻¹ and −682 g C m⁻² year⁻¹ for 2003 and 2004, respectively.     

Stable isotope labelling of earthworms can help deciphering belowground–aboveground interactions involving earthworms,mycorrhizal fungi,plants and aphids

Functional relationships between belowground detritivores and/or symbionts and aboveground primary producers and their herbivores are not well studied. In a factorial greenhouse experiment we studied interactions between earthworms (addition/no addition of Lumbricus terrestris; Clitellata: Lumbricidae) and arbuscular-mycorrhizal fungi (AMF; with/without inoculation of Glomus mosseae; Glomerales: Glomeraceae) on the leguminous herb Trifolium repens (Fabales: Fabaceae) and associated plant aphids (Aphis gossypii, A. craccivora; Hemiptera: Aphidoidea). In order to be able to trace organismic interactions, earthworms were dual-labelled with stable isotopes (¹⁵N-ammonium nitrate and ¹³C-glucose). We specifically wanted to investigate whether (i) isotopic signals can be traced from the labelled earthworms via surface castings, plant roots and leaves to plant aphids and (ii) these compartments differ in their incorporation of stable isotopes. Our results show that the tested organismic compartments differed significantly in their ¹⁵N isotope enrichments measured seven days after the introduction of earthworms. ¹⁵N isotope incorporation was highest in casts followed by earthworm tissue, roots and leaves, with lowest ¹⁵N signature in aphids. The ¹³C signal in roots, leaves and aphids was similar across all treatments and is for this reason not recommendable for tracing short-term interactions over multitrophic levels. AMF symbiosis affected stable isotope incorporation differently in different subsystems: the ¹⁵N isotope signature was higher below ground (in roots) but lower above ground (leaves and aphids) in AMF-inoculated mesocosms compared to AMF-free mesocosms (significant subsystem × AMF interaction). Aphid infestation was unaffected by AMF and/or earthworms. Generally, these results demonstrate that plants utilize nutrients excreted by earthworms and incorporate these nutrients into their roots, leaf tissue and phloem sap from where aphids suck. Hence, these results show that earthworms and plant aphids are functionally interlinked. Further, ¹⁵N-labelling earthworms may represent a promising tool to investigate nutrient uptake by plants and consequences for belowground-aboveground multitrophic interactions.     

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.     

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.