Dispersal patterns and behaviour of the nematode Phasmarhabditis hermaphrodita in mineral soils and organic media

The commercially available parasitic nematode Phasmarhabditis hermaphrodita is an effective biocontrol agent for slugs and particularly Deroceras reticulatum, a widespread pest species. Use of the nematode is currently limited by cost and it may be that by developing a fuller understanding of the ecology and behaviour of this nematode, more cost effective application strategies can be developed. We investigated the ability of two strains of P. hermaphrodita (one newly isolated and one that had been maintained in vitro for >15 years) to move through mineral soils and organic media. Active dispersal of both strains was found to be greatest in organic media (bark chips and leaf litter, and to a lesser extent peat) and the nematode was capable of growth and reproduction in leaf litter. Conversely, active dispersal was poor in mineral soils. Nematodes moved further in a clay loam compared with a sandy loam, and moved more at a bulk density of 1.0 vs. 1.2 Mg m⁻³. However, P. hermaphrodita was capable of moving greater distances in mineral soils by using the earthworm Lumbricus terrestris as a phoretic host. Our data suggest that P. hermaphrodita is a facultative parasite that is adapted to living in leaf litter and organic material where slugs frequently rest. The implications of these findings for using the nematode as a biological control agent for slugs are discussed.     

Successful analysis of gut contents in fungal-feeding oribatid mites by combining body-surface washing and PCR

The feeding habits of soil mesofauna have been a mystery for decades, and depending on the methods used, different degrees of feeding specialisation have been observed. A new way to study the almost unknown feeding habits of soil mesofauna e.g. oribatid mites is to use PCR-based techniques. When applying PCR on these small organisms, the low amount of ingested DNA can cause problems. Even more important is to certify that the amplified DNA does not originate from body-surface contamination. The aim of this study was to analyse if washing of the body surface combined with PCR can be a successful approach when identifying the food ingested by fungivorous mites. The method was developed in a laboratory system where we used the oribatid mite Archegozetes longisetosus as a model organism due to its relatively short life cycle and ease of laboratory culturing. The results demonstrated that surface contamination is a serious problem. Both washing and dissection was needed to remove surface contamination on such small organisms. To get a reliable result the samples also had to contain at least five pooled guts, but preferably ten. This is the first step towards a successful use of PCR-based methods to study natural feeding habits of species most likely contaminated on the body surface. When modified for field conditions, the results obtained by this method have a high potential to answer many questions about the animals feeding habits, and their functional role in the soil.     

Development and reproduction of Sancassania polyphyllae (Acari: Acaridae) feeding on entomopathogenic nematodes and tissues of insect larvae

We studied the effect of different food sources, infective juveniles of the entomopathogenic nematodes, Steinernema feltiae and Heterorhabditis bacteriophora (Rhabditida: Steinernematidae, Heterorhabditidae), and tissues from the insect larva, Polyphylla fullo (Coleoptera: Scarabaeidae) or Galleria mellonella (Lepidoptera: Pyralidae), on the development, reproduction and longevity of Sancassania polyphyllae (Acari: Acaridae). We showed that the immature mite stages - protonymph and tritonymph - could develop to the next developmental stage on living or sonicated (i.e., ruptured) S. feltiae or H. bacteriophora. However, the mite larval stage could only develop to the next developmental stage on sonicated infective juveniles of the nematodes. Subsequently, we demonstrated that S. polyphyllae completed development from protonymph to adult on live S. feltiae or H. bacteriophora, whereas all immature stages of S. polyphyllae completed their development from larva to adult on insect tissues. The total developmental period of S. polyphyllae that fed on insect tissues was significantly shorter than those that fed on live infective juveniles. The pre-oviposition, oviposition, and post-oviposition periods and female longevity were not significantly different among the food sources. The total and daily fecundity of S. polyphyllae feeding on P. fullo and G. mellonella was significantly higher than those feeding on S. feltiae and H. bacteriophora, although there was no significant difference observed between P. fullo and G. mellonella or between S. feltiae and H. bacteriophora. The net reproductive rate (R₀) was highest (588.3♀/♀) when S. polyphyllae fed on P. fullo. The longest mean generation time (T₀) occurred on H. bacteriophora (12.6 days) and the shortest occurred on P. fullo (10.5 days). S. polyphyllae, which fed on P. fullo (r_m=0.61) and G. mellonella (r_m=0.55) had the highest intrinsic rate of increase (r_m) compared to mites that fed on S .feltiae (r_m=0.45) and H. bacteriophora (r_m=0.41).     

Strong bottom-up and weak top-down effects in soil: nematode-parasitized insects and nematode-trapping fungi

The soils of the Bodega Marine Reserve (BMR, Sonoma County, California) contain many nematode-trapping fungi and many ghost moth larvae parasitized by entomopathogenic nematodes. The current study determined whether these nematode-parasitized moth larvae, which can produce very large numbers of nematodes, enhanced the population densities of nematode-trapping fungi and whether the fungi trapped substantial numbers of nematodes emerging and dispersing from moths. Wax moths were used in place of ghost moths because the former are easier to obtain. When nematode-parasitized moth larvae were added to laboratory microcosms containing BMR field soil, the population densities of four nematode-trapping fungi increased substantially. The greatest increase in population density was by Arthrobotrys oligospora, which uses adhesive networks to capture nematodes. A. oligospora population density increased about 10 times when the added moth larvae were parasitized by the nematode Heterorhabditis marelatus and about 100 times when added moth larvae were parasitized by the nematode Steinernema glaseri. Other trapping fungi endemic to the soil and enhanced by nematode-parasitized moth larvae included Myzocytium glutinosporum, Drechslerella brochopaga, and Gamsylella gephyropaga, which produce adhesive spores, constricting rings, and adhesive branches, respectively. The data suggest that the previously documented abundance and diversity of nematode-trapping fungi in BMR soil can be explained, at least in part, by nematode-parasitized insects, although that inference requires further studies with ghost moths. The strong bottom-up enhancement of nematode-trapping fungi was not matched by a strong top-down suppression of nematodes, i.e. the fungi trapped fewer than 30% of dispersing nematodes.     

Detecting ingested plant DNA in soil-living insect larvae

Although a significant proportion of plant tissue is located in roots and other below-ground parts of plants, little is known on the dietary choices of root-feeding insects. This is caused by a lack of adequate methodology which would allow tracking below-ground trophic interactions between insects and plants. Here, we present a DNA-based approach to examine this relationship. Feeding experiments were established where either wheat (Triticum aestivum) or maize (Zea mays) was fed to Agriotes larvae (Coleoptera: Elateridae), allowing them to digest for up to 72 h. Due to the very small amount of plant tissue ingested (max = 6.76 mg), DNA extraction procedures and the sensitivity of polymerase chain reaction (PCR) had to be optimized. Whole-body DNA extracts of larvae were tested for the presence of both rbcL and trnL plastid DNA using universal primers. Moreover, based on cpDNA sequences encoding chloroplast tRNA for leucine (trnL), specific primers for maize and wheat were developed. With both, general and specific primers, plant DNA was detectable in the guts of Agriotes larvae for up to 72 h post-feeding, the maximum time of digestion in these experiments. No significant effect of time since feeding on plant DNA detection success was observed, except for the specific primers in maize-fed larvae. Here, plant DNA detection was negatively correlated with the duration of digestion. Both, meal size and initial mass of the individual larvae did not affect the rate of larvae testing positive for plant DNA. The outcomes of this study represent a first step towards a specific analysis of the dietary choices of soil-living herbivores to further increase our understanding of animal-plant feeding interactions in the soil.     

The role of introduced mammals and inverse density-dependent predation in the conservation of Hutton's shearwater

This study investigated the impact of stoat Mustela erminea predation on the endangered Hutton's shearwater Puffinus huttoni. Breeding success of Hutton' shearwaters was significantly lower in two seasons of stoat control than in eight seasons with no control: suggesting that natural environmental variation has a greater influence on breeding success than stoat predation. Monitoring Hutton's shearwaters and mainland colonies of sooty shearwaters Puffinus griseus indicated that small colonies suffered higher predation and lower breeding success than large colonies, demonstrating that predation in these two species is inversely density dependent and explaining the low predation rates observed in the very large Hutton's shearwater colonies. The presence of another introduced predator, feral pigs Sus scrofa, within six extinct colonies of Hutton's shearwaters and at the boundaries of the two extant colonies, strongly suggests that this species was responsible for the historic contraction in breeding range. Controlling stoats within the two remaining colonies is unlikely to assist in the conservation of Hutton's shearwater. Conservation efforts would be better spent protecting the two remaining colonies from pigs and in trying to establish new breeding sites.     

Influence of irrigation on the distribution and control of the nematode Meloidogyne javanica by the biocontrol bacterium Pasteuria penetrans in the field

Pasteuria penetrans, a bacterial parasite of plant-parasitic nematodes, is used to control root-knot nematode Meloidogyne spp. populations in vegetable crops. But its efficiency is variable, mostly because of the patchy distribution of the bacteria in arable fields. As the infective P. penetrans are non-motile bacteria in soil, abiotic soil factors can affect the bacteria–nematode relationships. An epidemiological study, conducted in a vegetable field, showed that abiotic factors such as irrigation, soil water holding capacity and texture, affected the efficiency of P. penetrans. A correspondence analysis between these abiotic factors and the density of P. penetrans spores in the soil, and the proportion of Meloidogyne javanica juveniles infected by the bacteria, revealed that irrigation affected directly the distribution of the spores in soil pores related to their passive transport by water flow. Laboratory experiments conducted on the passive transport of spores confirmed that intensive irrigation leached the spores down the soil profile and decreased the percentage of infected Meloidogyne juveniles.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.     

The effect of soil water flow and soil properties on the motility of second-stage juveniles of the root-knot nematode (Meloidogyne incognita)

The movement of root-knot nematode Meloidogyne incognita second-stage juveniles was investigated in a column filled with two types of fillings, andisol or sand, under a saturated condition. After incubation with or without soil water flow, the changing distribution of nematodes was monitored. As a result, we observed a tendency for different movement traits of nematodes between andisol and sand. The distance of nematodes movement in sand was always greater than that in andisol in both a percolation test and non-percolation test. In the percolation test, distance of nematodes movement in sand gradually began to increase with increasing time. We attribute this result to a gradual decrease in nematodes activity with time. Nematodes were always affected by the water flow rate, but the distance of nematodes movement did not correspond to water flow volume. We also observed that mobility of nematodes in sand were always greater than that in andisol at both percolation test and non-percolation test. These results are attributed to differences in soil structure and pore distribution of andisol and sand. This study demonstrates a difference in the movement of nematodes influenced by water flow. However, this study also presents evidence that nematodes can resist soil water flow rates that are possible in actual field conditions. Moreover, mobility of nematodes changed considerably with soil structure or pore distribution. All these may contribute to a more stable positioning of nematodes around the rhizosphere of their host plants, and therefore increase the possibility of successful parasitization.     

Peat amendment and production of different crop plants affect earthworm populations in field soil

A field experiment was conducted to study the effects of peat amendment and crop production system on earthworms. The experiment was established on a field previously cultivated with oats and with silt as the main soil type. Perennial crops strawberry, timothy and caraway, and annual crops rye, turnip rape, buckwheat, onion and fiddleneck were cultivated with conventional methods. All the crops were grown with and without soil amendment with peat. Earthworms were sampled twice: 4 and 28 months after establishment of the experiment. In the former case part of the experimental plots were soil sampled and hand sorted for estimation of earthworms. In the latter case all experimental plots were sampled and both soil sampling and mustard extraction was carried out. Soil organic carbon and microbial biomass was measured at 14 and 28 months. Peat increased the abundance of juvenile Aporrectodea caliginosa by 74% in three growing seasons, but had no effect on adult numbers. Lumbricus terrestris numbers were not increased by peat treatment. Three season cultivation of caraway favoured both A. caliginosa and L. terrestris. An equal abundance of A. caliginosa was also found in plots cultivated with turnip rape and fiddleneck. Total earthworm and especially A. caliginosa numbers were very small in plastic-mulched strawberry beds. This was mainly attributed to repeated use of the insecticide endosulfan. With the strawberry plots omitted there was a significant correlation between soil microbial N measured at 14 months and juvenile Aporrectodea spp. and Lumbricus spp. numbers measured at 28 months. Adult earthworm numbers were not associated with either soil organic C or microbial biomass.     

Nest predation in a fragmented Afrotropical forest: evidence from natural and artificial nests

Nest predation accounts for a substantial share of nest failure and low reproductive success in most tropical songbirds. Normally, forest fragmentation leads to an increase in nest predation pressure due to reduced cover, fewer (and poorer) nest sites and predator influxes from the surrounding habitats. To test this hypothesis, we studied natural nesting behaviour and nest success of the white-starred robin (Pogonocichla stellata) in seven Afrotropical forest fragments differing in size and level of habitat disturbance. Based on data from 12 nests, we estimate that 29% of all natural nests initiated by the robins survive to produce fledglings across all fragments. We also conducted an experiment using artificial (plasticine) model-eggs to reveal potential predators and compare relative predation rates amongst fragments. This experiment revealed that small mammals might be the major predators on robin nests at the egg-stage. In addition, it showed that the highest incidences of nest disturbance during this stage were in the most heavily disturbed fragment. This was presumably attributable to an influx of mammalian predators from the surrounding habitats as forest degradation created suitable habitats for them. Such an infiltration was recently reported in this study site. Both nest placement and microhabitat did not significantly affect depredation levels in our experiment. This suggests that depredation was predominantly incidental (i.e., predators mainly encountered nests fortuitously while foraging for other food items), where the likelihood of encountering a nest largely depended upon the prevalence of the principal potential predators - the small mammals.     

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.     

Molecular markers and ex situ conservation of the European elms (Ulmus spp.)

The enormous losses suffered by the European elms during recent Dutch elm disease outbreaks led to concern over the conservation of elm genetic resources, and the subsequent establishment of a series of ex situ collections. However, as ex situ collections are inevitably finite in size, some consideration needs to be given to selecting which samples to include in them. To contribute towards this process for European ex situ elm collections we have undertaken genetic studies on a Europe-wide sample of 535 individuals. A major aim has been to use genetic markers to clarify the identification of samples to ensure that the ex situ collections contain a representative spread of taxonomic diversity. This is important given the paucity of mature elms in the landscape due to Dutch elm disease. The lack of mature material (critical for identification) compounds identification problems in what was already a taxonomically difficult group. Our data (derived from random amplified polymorphic DNA and inter-simple sequence repeats) have provided a useful supplement to morphology in undertaking such sample identifications. The molecular data served to highlight mis-identified samples and led to extensive revisions of sample identities within individual countries. Our results were less useful in detecting regional intra-specific genetic structure, and do not provide sufficient information for prioritising within-species sample selections.     

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

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

Molecular phylogenetic analysis of a novel strain from Neelipleona enriches Wolbachia diversity in soil biota

Eubacterial cellular endoparasites belonging to the genus Wolbachia (Rickettsiales) are extremely widespread symbionts of Arthropoda and Nematoda. Their ability to manipulate the reproductive behavior of the host is of particular importance to the fauna of the deep soil horizon, an environment in which parthenogenesis-inducing symbionts can play a crucial role in shaping population dynamics and speciation processes. In this study, three novel cases of infection in parthenogenetic Collembola (Parisotoma notabilis, Neelus murinus and Megalothorax minimus) are described. Sequences for molecular markers 16S rDNA and ftsZ were obtained for each species; their phylogenetic affinities with known Wolbachia supergroups were established using Bayesian inference. The analysis confirmed the presence of a Wolbachia strain belonging to the supergroup E, already reported from Folsomia candida and the Tullbergiidae, in the isotomid P. notabilis, while the Neelipleona M. minimus and N. murinus host a well differentiated strain which is phylogenetically distinct from supergroup E. Multiple events of Wolbachia infection in springtails as well as a richer diversity of the symbiont strains in soil arthropods were hereby confirmed.     

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

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

Long-term soil organic carbon dynamics in a subhumid tropical climate: C data in mixed C3/C4 cropping and modeling with ROTHC

Scanty information on long-term soil organic carbon (SOC) dynamics hampers validation of SOC models in the tropics. We observed SOC content changes in a 16-year continuously cropped agroforestry experiment in Ibadan, south-western Nigeria. SOC levels declined in all treatments. The decline was most pronounced in the no-tree control treatments with continuous maize and cowpea cropping, where SOC levels dropped from the initial 15.4 to 7.3-8.0 Mg C ha⁻¹ in the 0-12 cm topsoil in 16 years. In the two continuously cropped alley cropping (AC) systems, one with Leucaena leucocephala and one with Senna siamea trees, SOC levels dropped to 10.7-13.2 Mg C ha⁻¹. Compared to the no-tree control treatments, an annual application of an additional 8.5 Mg ha⁻¹ (dry matter) of plant residues, mainly tree prunings, led to an extra 3.5 Mg C ha⁻¹ (∼0.2% C) in the 0-12 cm top soil after 11 years, and 4.1 Mg C ha⁻¹ after 16 years. The addition of NPK fertilizer had little effect on the quantities of above-ground plant residues returned to the soil, and there was no evidence that the fertilizer affected the rate of SOC decomposition. The fact that both C₃ and C₄ plants returned organic matter to the soil in all cropping systems, but in contrasting proportions, led to clear contrasts in the ¹³C abundance in the SOC. This ¹³C information, together with the measured SOC contents, was used to test the ROTHC model. Decomposition was very fast, illustrated by the fact that we had to double all decomposition rate constants in the model in order to simulate the measured contrasts in SOC contents and δ¹³C between the AC treatments and the no-tree controls. We hypothesized (1) that the pruning materials from the legume trees and/or the extra rhizodeposition from the tree roots in the AC treatments accelerated the decomposition of the SOC present at the start of the experiment (true C-priming), and/or (2) that the physical protection of microbial biomass and metabolites by the clay fraction on this site, having a sandy top soil in which clay minerals are mainly of the 1:1 type, is lower than assumed by the model.