Culture of commercially obtained Lumbricus terrestris L.: Implications for sub-lethal ecotoxicological testing

The use of commercially purchased or field-collected earthworms of unknown age, exposure or pre-treatment in sub-lethal ecotoxicological studies is questionable. In this study, adult (clitellate) Lumbricus terrestris, obtained from 5 commercial suppliers in the UK and also field collected, were kept under controlled environmental conditions (15 °C and 24 h darkness) in a sterilised loam soil and fed horse manure. Survival, biomass and cocoon production was monitored every 4 weeks over 1 y. Marked differences were recorded in survival rates (ranging from 40-100% after 40 weeks) and cocoon production (ranging from 15.1 to 32.2 worm⁻¹ y⁻¹) between treatments. Biomass in all treatments (mean mass 4.32-5.61 g at the outset) increased with time to week 20 (maximum 6.7 g) and then declined steadily (3.23-4.7 g at week 52). This pattern was also observed in cocoon production and was considered to be a function of an initial period of acclimation (0-12 weeks) followed by a period of high production (12-36 weeks) under optimal conditions and then fatigue (36-52 weeks) caused by reproductive exhaustion. Results suggest that earthworm origin may influence the validity and reproducibility of sub-lethal ecotoxicological studies and where applicable laboratory-reared earthworms of known age and history are recommended as test subjects.     

Field investigations of Lumbricus terrestris spatial distribution and dispersal through monitoring of manipulated, enclosed plots

A field experiment in managed woodland was set up to examine the effects of manipulated population density and resource availability on spatial distribution and dispersal of the anecic earthworm Lumbricus terrestris. Experiments over 2 years, made use of 1 m² field enclosures with associated trapping units to assess emigration rates at control and enhanced L. terrestris densities and different levels of leaf litter availability. Densities were manipulated twice; at the outset and again after 1 year when visually tagged animals obtained from 2 origins were introduced. Population density had a significant effect on dispersal (p < 0.01, p < 0.05 in Year 1 and Year 2 respectively) with more captures (pro rata) at the higher density compared with controls over the experimental period. Food availability only had a significant effect during the initial week of the experiment. L. terrestris midden arrangement was found to be regular across 1 m² plots and regularity increased with an increase in midden number. Mean (±S.E.) midden number was 30.34 ± 0.77 m⁻² and 28.06 ± 0.5 m⁻², during the first and second year of the experiment respectively and this was unaffected by additions. Inter-midden distance was recorded at 0.13 ± 0.0014 m. Results suggest that L. terrestris dispersal can be affected by population density and resource availability.     

Life history characteristics of Lumbricus terrestris and effects of the veterinary antiparasitic compounds ivermectin and fenbendazole

Earthworms in pastureland may be exposed to residues of veterinary medicines excreted in the faeces of treated cattle. Possible sub-lethal effects of these drugs on life history characteristics are critical to earthworm populations, but there are no internationally accepted guidelines for the laboratory testing that is essential as a supplement to field studies. In the laboratory, Lumbricus terrestris were kept in artificial soil and offered dung collected from cattle with sustained-release boluses of ivermectin or fenbendazole, or from untreated cattle (control). Earthworms were therefore exposed to drug residues in concentrations excreted naturally by treated cattle. Furthermore, worms were exposed to the drug in the natural way, i.e. by ingestion. A matrix model based on survival and reproductive rates as functions of life-stages was used to quantify the population dynamics of L. terrestris. There were no adverse effects of either ivermectin or fenbendazole on the individual survival and growth of L. terrestris. Mature worms produced on average 2-3 cocoons per worm per month irrespective of drug treatment, and about 50% of these cocoons hatched successfully. The median time to hatching (incubation time) depended on the age of the parent worms and was significantly lower for cocoons in the ivermectin group compared to controls, as the worms became older. The incubation time varied considerably in all groups; even within a batch of cocoons produced during 1 month by one pair of worms, the period sometimes varied by more than 1 year. The intrinsic rate of natural increase (r≈0.0044 d⁻¹) was not affected by either drug treatment. The methods and the model developed in this study are suitable for long-term studies on L. terrestris and give ecologically relevant information on population dynamics. There were no negative effects of ivermectin and fenbendazole on life history characteristics of L. terrestris and it is unlikely that earthworm populations will be affected in the field following normal use of sustained-release boluses in cattle. Finally, the matrix model points to adult survival rate and cocoon viability as the most important variables to be included in future ecotoxicological tests on L. terrestris.     

Growth and cocoon production of Drawidia nepalensis (Oligochaeta)

Summary The biology of the vermicomposting species, Drawidia nepalensis Mich. is poorly understood. We therefore studied the growth of this species at 25°C in urine-free cow and horse manure. The rate of maturation and cocoon production were studied under conditions of favourable moisture and nutrition over a period of 240 days. The results showed that D. nepalensis grew relatively slowly in comparison to other vermicomposting species, and that copulation was not a prerequisite for the production of viable cocoons, indicating that D. nepalensis may be parthenogenetic. A faster growth rate, and the production of more cocoons in cow compared with horse manure indicate that cow manure is a preferred source of food for D. nepalensis.     

Worms from the cold: Lumbricid life stages in boreal clay during frost

The vertical distribution and activity of earthworm life stages were studied in an arable field during 0.5 m deep frost. The anecic Lumbricus terrestris L. were below the frost at the bottom of their home burrows (max. depth 1.0 m) and remained there apparently active. Their burrows were open, free of ice and water. The endogeic Aporrectodea caliginosa Sav., mainly small juveniles, were aestivating in the frost layer, which confirms freeze-tolerance in this species. Large A. caliginosa individuals were actively burrowing below the frost down to 1 m depth at soil temperatures close to +1 °C, frost evidently triggering much deeper burrowing than summer droughts. Demonstrating cold-hardiness, viable cocoons of both A. caliginosa and L. terrestris were obtained within a 0-0.25 m layer, frozen for ca. one month prior to sampling. These two common earthworms of boreal soils seem to over-winter in all life stages and remain active below the frost, potentially contributing to the maintenance of subsoil processes during the winter months.     

Growth and cocoon production by the earthworm Metaphire houletti (Oligochaeta) in different food sources

 Quantitative observations on the biology of Metaphire houletti were made in cow and horse manure and oak litter in laboratory conditions over a period of 240 days. The study revealed that copulation is not a prerequisite for production of viable cocoons, indicating that M. houletti may be parthenogenetic. The mean growth rate was 2.86 mg worm^–1 day^–1 reaching sexual maturity at 45 days and producing 0.015 cocoons worm^–1 day^–1 (kept singly) and 2.82 mg worm^–1 day^–1, reaching sexual maturity at 45 days and producing 0.03 cocoons worm^–1 day^–1 (kept in batches) in cow manure; 4.08 mg worm^–1 day^–1, reaching sexual maturity at 46 days and producing 0.02 cocoons worm^–1 day^–1 (kept singly) and 2.97 mg worm^–1 day^–1, reaching sexual maturity at 45 days and producing 0.016 cocoons worm^–1 day^–1 (kept in batches) in horse manure; 3.73 mg worm^–1 day^–1 reaching sexual maturity at 45 day and producing 0.023 cocoons worm^–1 day^–1 (kept singly) and 2.73 mg worm^–1 day^–1, reaching sexual maturity at 47 days and producing 0.028 cocoons worm^–1 day^–1 (kept in batches) in oak litter. After an incubation period of 31.9±1.2 days 82% of the cocoons hatched with a mean of 1.12±0.06 hatchlings per cocoon. The earthworms reared in batches did not demonstrate any advantage over those reared singly in all substrates. Higher growth rates were observed in earthworms raised singly than those raised in batches in all substrates. Received: 30 April 1998     

Reproduction and growth of three deep-burrowing earthworms (Lumbricidae) in laboratory culture in order to assess production for soil restoration

Laboratory experiments were conducted to examine the growth and reproduction of three deep-burrowing lumbricids, Aporrectodea longa, Lumbricus terrestris, and Octolasion cyaneum. The reproductive output was recorded as 18.8, 38.0, and 32.3 cocoons per worm per year for A. longa, L. terrestris, and O. cyaneum, respectively. For the same species, maturity was reached at a mean mass of 3.9, 5.0 and 2.4 g, within 3 months from the hatchling stage by L. terrestris and within 4 months by the other two species. The hatching success of cocoons at 15 and 20°C was within the range of 70–80% for each species, except A. longa at the higher temperature, where a viability of 47% was recorded. Twenty percent of viable O. cyaneum cocoons produced twin hatchlings, compared with only one percent for A. longa and L. terrestris. A combination of these results suggests that a complete life-cycle for each species could be achieved within 6 months (L. terrestris and A. longa) or 7–8 months (O. cyaneum). Each species has particular life-cycle strategies that would aid survival and colonisation, under field conditions, if inoculated into restored soils.     

Long-term fertilization and manuring effects on physically-separated soil organic matter pools under a wheat-wheat-maize cropping system in an arid region of China

With increasing food demand worldwide, agriculture in semiarid and arid regions becomes increasingly important, though knowledge about organic matter (OM) conserving management systems is scarce. This study aimed at examining organic C (OC) and nitrogen (N) concentrations in various soil OM pools affected by 26-years application of chemical fertilizer and farmyard manure at an arid site of Gansu Province, China. Macro OM (>0.05 mm) was extracted by wet sieving and then separated into light macro OM (<1.8 g cm⁻³) and heavy macro OM (>1.8 g cm⁻³) sub-fractions; bulk soil was differentiated into free particulate OM (FPOM, <1.6 g cm⁻³), occluded particulate OM (OPOM, 1.6-1.8 g cm⁻³) and mineral-associated OM (>1.8 g cm⁻³). OC and N concentrations of heavy macro OM and FPOM were slightly affected by long-term N fertilization alone and its combination with P and K, but their magnitudes of change had not significantly contributed to total soil OC and N concentrations. Farmyard manure increased light macro OC and N by 58 and 70%, heavy macro OC and N by 86 and 117%, free particulate OC and N by 29 and 55%, occluded particulate OC and N by 29 and 55%, and mineral-associated OC and N by 44 and 48%, respectively, compared to nil-manure. Mineral fertilization improved soil OM quality by decreasing C/N ratio in the light macro OM and FPOM fractions where farmyard manure was absent. Organic manure led to a decline of the C/N ratio in all physically-separated OM fractions possibly due to the increased input of processed organic materials. We found about two thirds of macro OM was actually located within 2-0.05 mm organo-mineral associations or/and aggregates. In conclusion, this study stresses the vital importance to apply organic manure to the wheat-corn production system characterized by straw removal and conventional tillage in the region.     

Depth of cocoon deposition by three earthworm species in mesocosms

Earthworms were maintained in two types of soil-filled mesocosm. Type 1, designed for use in soil-inoculation studies, was only 0.15 m deep. Sampling revealed the position at which cocoons were deposited by earthworms in mono-species culture. Whilst adequate for shallow-working worms, larger species may have experienced restricted burrow formation and associated cocoon deposition. Therefore, Type 2 mesocosms (1.0 m deep) were also used. Here, earthworms were found to burrow throughout the soil columns, but cocoons were mainly deposited within 0.25 m of the soil surface (95% overall). The deepest cocoon deposition was at 0.4 m by Lumbricus terrestris, although 45% of the cocoons for this species were located in the upper 0.05 m of the soil, compared with 70% and 71% for Aporrectodea longa and Octolasion cyaneum, respectively. Comparisons between mesocosms showed that their depth affected cocoon distribution in the soil and that differences were also present compared with field-collected results. Reasons for this are discussed, as are implications for soil inoculation with earthworms. If cocoons are viewed as a potential inoculum for soil restoration work, their harvesting and spreading in soil may assist successful colonisation.     

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.     

Inoculation of root microorganisms for sustainable wheat-rice and wheat-black gram rotations in India

The scarcity of non-renewable resources such as soils and fertilizers and the consequences of climate change can dramatically influence the food security of future generations. Mutualistic root microorganisms such as plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) can improve plant fitness. We tested the growth response of wheat (Triticum aestivum [L.]), rice (Oriza sativa [L.]) and black gram (Vigna mungo [L.], Hepper) to an inoculation of AMF and PGPR alone or in combination over two years at seven locations in a region extending from the Himalayan foothills to the Indo-Gangetic plain. The AMF applied consisted of a consortium of different strains, the PGPR of two fluorescent Pseudomonas strains (Pseudomonas jessenii, R62; Pseudomonas synxantha, R81), derived from wheat rhizosphere from one test region. We found that dual inoculation of wheat with PGPR and AMF increased grain yield by 41% as compared to un-inoculated controls. Yield responses to the inoculants were highest at locations with previously low yields. AMF or PGPR alone augmented wheat grain yield by 29% and 31%, respectively. The bio-inoculants were effective both at Zero and at farmers’ practice fertilization level (70 kg N ha⁻¹, 11 kg P ha⁻¹ in mineral form to wheat crop). Also raw protein (nitrogen × 5.7) and mineral nutrient concentration of wheat grains (phosphorus, potassium, copper, iron, zinc, manganese) were higher after inoculation (+6% to +53%). Phosphorus use efficiency of wheat grains [kg P grain kg⁻¹ P fertilizer] was increased by 95%. AMF and PGPR application also improved soil quality as indicated by increased soil enzyme activities of alkaline and acid phosphatase, urease and dehydrogenase. Effects on rice and black gram yields were far less pronounced over two cropping seasons, suggesting that AMF and PGPR isolated from the target crop were more efficient. We conclude that mutualistic root microorganisms have a high potential for contributing to food security and for improving nutrition status in southern countries, while safeguarding natural resources such as P stocks.     

Cultivation of transgenic cyanophycin-producing potatoes does not negatively affect growth,reproduction and activity of the earthworm Lumbricus terrestris (L.)

A microcosm experiment was performed to investigate the effects of post-harvest potato tubers from transgenic cyanophycin-producing potatoes on Lumbricus terrestris (L.) activity and biomass, number of cocoons and their hatchability as well as the remaining cyanophycin content in soil and cast samples during a period of 80 days. Potato tubers from four transgenic potato events with different cyanophycin content in a range from 0.8 to 7.5% were compared to the near isogenic, non-transgenic control (Solanum tuberosum L. cv. Albatros) and a comparative potato cultivar (S. tuberosum L. cv. Désirée). One treatment with transgenic tuber residue but without earthworms was prepared as an additional control. Potato tuber loss from the surface of the microcosms was significantly higher in the treatments with transgenic potato tubers compared with non-transgenic treatments. It can be estimated that the earthworm contribution to potato tuber loss from the soil surface was approximately 61%. Mean number of cocoons in addition to the number of hatched cocoons varied from 2.6 to 6.2 and from 7 to 15 accounting for 45.2–83.35% hatchability, respectively, but no significant differences between the treatments were found. The same was true for the development of earthworm biomass in the various treatments. The cyanophycin content in soil samples was significantly higher when earthworms were present indicating that the cyanophycin content in the upper soil layer might have been enhanced through earthworm burrowing activity. Overall, it is concluded that tubers from transgenic cyanophycin potatoes are easily degradable and neither inhibit nor stimulate earthworm growth, reproduction, and activity.     

Internal regulation of reproduction seasonality in earthworm Dendrobaena octaedra (Savigny, 1826) (Lumbricidae, Oligochaeta)

The study was conducted on Dendrobaena octaedra—a small epigeic earthworm species common in different forest types. In the laboratory the P (parent animals collected in forest) and F1 generations were cultured separately in containers filled with 50 g artificial soil. The containers were kept at 15±0.5 °C, 80% humidity and constant artificial light of low intensity. Every month during the 47 months of culture, the animals and cocoons were removed from the soil by washing on a sieve, weighed, and replaced into new soil. Individuals of the F1 generation did not reproduce continuously. Cocoon production was seasonal, despite culture in constant conditions throughout the whole experiment. Reproduction was highest in spring and summer, and dropped in the winter months. Seasonality characterized the fraction of animals reproducing as well as the number of cocoons produced. The observed seasonal changes in the cocoon production of the F1 generation cultured in constant conditions suggest that internal regulation of reproduction may exist in the earthworm D. octaedra. External factors like temperature, moisture, photoperiod or food supply, which could be responsible for seasonality of reproduction were missing.     

Miscanthus sinensis and wild flowers as food resources of Lumbricus terrestris L.

Senescent leaves of Miscanthus sinensis contained 36% soluble polysaccharides, 26% cellulose and had a C/N ratio of 45. In 11 wild flower species contents of soluble polysaccharides (21–30%), cellulose (3–16%) and C/N ratio (13–31) were lower. Decomposing leaves of M. sinensis lost weight at a rate of 0.002 day⁻¹, increased the C/N ratio from 45 to about 100, the bacterial biomass from 0.4 to 1 μg C mg⁻¹ dry weight, and decreased the tensile strength from 35 to 10 N. The withdrawal rate of Lumbricus terrestris with senescent leaves of M. sinensis was 30 mg g⁻¹ week⁻¹; the feeding rate was lower. With most senescent wild flowers withdrawal and feeding rates were higher. During decomposition of M. sinensis withdrawal rates increased to about 90, and feeding rates to about 30 mg g⁻¹ week⁻¹. The rates were not related to soluble polysaccharides, cellulose, acid-insoluble residue, C/N ratio and the presence of trichomes on the leaves. The abundance of L. terrestris decreased in a meadow turned into a field of M. sinensis from 55 to 26 earthworms m⁻² and increased in a rotational maize field turned into wild flower strips from 28 to 46 earthworms m⁻². The species richness of earthworms decreased with M. sinensis from 7.2 to 4.7 and increased with wild flowers from 4.7 to 6.7 species per sampling unit.     

Life cycle of Pontoscolex corethrurus () in tropical artificial soil

Pontoscolex corethrurus (Müller) is a widespread, peregrine earthworm species of the Glossoscolecidae family, native to the Neotropics. This endogeic earthworm shows wide tolerance to environmental variations, being found in many different habitats and soil types throughout the tropics and sub-tropics. To evaluate the life cycle of this species in tropical artificial soil (TAS), a substrate used in ecotoxicological tests, and the influence of food availability and humidity on its growth, earthworms were incubated individually under laboratory conditions. The life cycle was evaluated in four treatments with 24 earthworms (replicates) each: TAS (120 g) without additional food (TAS0), TAS with 5 g of horse manure (TAS5), TAS with 10 g of manure (TAS10) and TAS with 25% greater soil moisture with 5 g of manure (TAS5H). Food was provided every 14 d and the containers maintained at room temperature (20 ± 4 °C). Cocoons collected in the field were placed individually in the different treatments and the hatching date, growth, development, and reproduction followed for 50 weeks after hatching. New cocoons hatched at 34 d, adulthood was reached at 8 months, and cocoon deposition began 3 months later, so that its life cycle was completed in 12 months. By the 13th week all juveniles in TAS0 had died due to insufficient food resources, while in the other treatments, all the animals had matured by 44 weeks, with significantly higher biomass and length in TAS10 (0.72 g, 5.4 cm), compared with TAS5 and TAS5H (0.59–0.61 g, 5.1–5.4 cm). Growth and weight gain were positively related to food availability, and no negative effect of excess moisture was observed on their growth. With additional food TAS showed no limitations for the development of the species, and it can be used for ecotoxicological tests involving acute toxicity (mortality) and avoidance, although its potential use in chronic tests (reproduction) is more limited. The length of its life cycle and other biological characteristics associated with this parthenogenetic species create some challenges that must be dealt with before this species can be recommended for wider use as a standard ecotoxicological test species.     

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.     

Endogeic and anecic earthworm abundance in six Midwestern cropping systems

Endogeic and juvenile anecic earthworm abundance was measured in soil samples and anecic populations were studied by counting midden numbers at the sites of two long-term cropping systems trials in South-central Wisconsin. The three grain and three forage systems at each site were designed to reflect a range of Midwestern USA production strategies. The primary objectives of this work were to determine if the abundance of endogeic or anecic earthworms varied among cropping systems or crop phases within a cropping system and were there specific management practices that impacted endogeic or anecic earthworm numbers. The earthworms present in the surface soil were: Aporrectodea tuberculata (Eisen), A. caliginosa (Savigny), A. trapezoides (Dugés); and juvenile Lumbricus terrestris (L.). True endogeic abundance was greatest in rotationally grazed pasture [188 m^?2 at Arlington (ARL) and 299 m^?2 at Elkhorn (ELK)], and smallest in conventional continuous corn (27 m^?2 at ARL and 32 m^?2 at ELK). The only type of anecic earthworm found was L. terrestris L. There was an average of 1.2 middens per adult anecic earthworm and the population of anecics was greatest in the no-till cash grain system (28 middens m^?2 at ARL, 18 m^?2 at ELK) and smallest in the conventional continuous corn system (3 middens m^?2 at ARL, 1 m^?2 at ELK). Earthworm numbers in individual crop phases within a cropping system were too variable from year-to-year to recommend using a single phase to characterize a whole cropping system. Indices for five management factors (tillage, manure inputs, solid stand, pesticide use, and crop diversity) were examined, and manure use and tillage were the most important impacting earthworm numbers across the range of cropping systems. Manure use was the most important management factor affecting endogeic earthworm numbers; but no-tillage was the most important for the juvenile and adult anecic groups and had a significantly positive influence on endogeic earthworm counts as well. The pesticides used, which were among the most commonly applied pesticides in the Midwestern USA, and increasing crop diversity did not have a significant effect on either the endogeic or anecic earthworm groups in this study. Consequently, designing cropping systems that reduce tillage and include manure with less regard to omitting pesticides or increasing crop diversity should enhance earthworm populations and probably improve sustainability.     

Litter- and ecosystem-driven decomposition under elevated CO2 and enhanced N deposition in a Sphagnum peatland

Peatlands represent massive global C pools and sinks. Carbon accumulation depends on the ratio between net primary production and decomposition, both of which can change under projected increases of atmospheric CO₂ and N deposition. The decomposition of litter is influenced by 1) the quality of the litter, and 2) the microenvironmental conditions in which the litter decomposes. This study aims at experimentally testing the effects of these two drivers in the context of global change. We studied the in situ litter decomposition from three common peatland species (Eriophorum vaginatum, Polytrichum strictum and Sphagnum fallax) collected after one year of litter production under pre-treatment conditions (elevated CO₂: 560 ppm or enhanced N: 3 g m⁻² y⁻¹ NH₄NO₃) and decomposed the following year under treatment conditions (same as pre-treatment). By considering the cross-effects between pre-treatments and treatments, we distinguished between the effects on mass loss of 1) the pre-treatment-induced litter quality and 2) the treatment conditions under which the litters were decomposing. The combination between CO₂ pre-treatment and CO₂ treatment reduced Polytrichum decomposition by −24% and this can be explained by litter quality-driven decomposition changes brought by the pre-treatment. CO₂ pre-treatment reduced Eriophorum litter quality, although this was not sufficient to predict decomposition. The N addition pre-treatment reduced the decomposition of Eriophorum, due to enhanced lignin and soluble phenols concentrations in the initial litter, and reduced litter-driven losses of starch and enhanced litter-driven losses of soluble phenols. While decomposition indices based on initial litter quality provide a broad explanation of quantitative and qualitative decomposition, they can only be taken as first approximations. Indeed, the microbial ATP activity, the litter N loss and resulting litter quality, were strongly altered irrespective of the compounds' initial concentration and by means of processes that occurred independently of the initial litter-qualitative changes. The experimental design was valuable to assess litter- and ecosystem-driven decomposition pathways simultaneously or independently. The ability to separate these two drivers makes it possible to attest the presence of litter-qualitative changes even without any litter biochemical determinations, and shows the screening potential of this approach for future experiments dealing with multiple plant species.     

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.     

Nitrous oxide emissions from a bermudagrass pasture: Interseeded winter rye and poultry litter

Adequate use of manure in grasslands may constitute an economical means of manure disposal and an abundant source of nutrients for plants; however, excessive nitrogen (N) additions to these soils could create new environmental risks such as increasing nitrous oxide (N₂O) emissions. These potentially adverse effects in grasslands may be mitigated by improved management practices. In pasture systems, the combined effects of poultry litter applications and interseeded rye (Secale cereale L.) on N₂O emissions are still not well established. This study was conducted to estimate the magnitude of soil surface N₂O fluxes as affected by interseeded winter rye forage, annually spring-applied composted turkey litter as well as by weather and soil parameters. Fluxes were measured by vented chambers during 2 yr in a bermudagrass (Cynodon dactylon [L.] Pers.) pasture in moderately well-drained Tonti gravelly silt loam (fine-loamy, active, mesic Typic Fragiudault) located in northwestern Arkansas, USA. During the 60 d following turkey litter applications, N₂O fluxes were frequently well correlated with soil nitrate (NO₃⁻; r: up to 0.82, P's < 0.05) implying substrate stimulation on soil N₂O production. Likewise, rainfall patterns strongly influenced N₂O fluxes. Large rainfalls of 91 and 32 mm occurred within 6 d prior to the maximum N₂O flux means (263 and 290 μg N m⁻² h⁻¹, respectively). Treatment effects on N₂O emissions were significant only in spring periods following manure addition, particularly in the second year of our study. In the spring of 2000, additions of composted turkey litter resulted in 1.5-fold increase in seasonal cumulative N₂O emissions (P = 0.04) which was directly associated to a numerically greater soil NO₃⁻. In the spring of 2001, soils planted to rye exhibited a pronounced significant effect on mitigating N₂O emissions (30 vs. 112 mg N m⁻²; P = 0.04). During the winter and early spring, rye growth also decreased quantities of both soil NO₃⁻ and water-filled pore space (WFPS) partly accounting for the lower N₂O emissions in these fields. These results suggest that because poultry litter additions increased and interseeded rye diminished N₂O emissions, the combined implementation of both management practices can produce environmental benefits while sustaining productivity in temperate pasture systems.