Metal soil pollution differentially affects both the behaviour and exposure of <Emphasis Type="Italic">A. caliginosa</Emphasis> and <Emphasis Type="Italic">L. terrestris</Emphasis>: a mesocosm study

The effects on two earthworm species of a gradient of metal contamination in soil collected close to a 50-year-old lead recycling factory were investigated in mesocosms filled with soil sampled at three distances from the factory (10, 30 and 60 m). After 5 weeks of exposure, earthworm litter consumption and weight change were measured. Burrow systems were analysed using X-ray tomography, and water infiltration was measured. No significant differences in earthworm weight or activity were observed between mesocosms filled with soil from 30 and 60 m. In contrast, both earthworm species significantly lost weight and burrowed less in the soil sampled at 10 m. In the cores filled with the soil collected at 10-m distance, Aporrectodea caliginosa avoided the highly contaminated first layer (0–5 cm) and burrowed deeper whereas Lumbricus terrestris burrowed relatively more in this layer. We assume that these different reactions are associated with their ecological types. Epi-anecic earthworms forage litter at the soil surface, whereas endogeic earthworms are geophagous and thus are able to forage deeper. This was further corroborated by the bioaccumulation factors measured for each species: for L. terrestris, BAF values for Pb and Cd only decreased slightly in the 10-m soil correlating with their overall reduced activity. However, BAF values for A. caliginosa were 20-fold lower compared to those observed in soil from 30 and 60 m. These modifications in burrowing behaviour in the 10-m mesocosms resulted in a significant and marked decrease in water infiltration rates but only for L. terrestris.     

Dehydration does not affect the radial pressures produced by the earthworm <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis>

As a soil dries, the earthworms in that soil dehydrate and become less active. Moisture stress may weaken an earthworm, lowering the radial pressure that the animal can produce. This possibility was investigated for the earthworm Aporrectodea caliginosa (Savigny). Pressures were compared for saturated earthworms (worms taken from saturated soil) and stressed earthworms (worms that had been partially dehydrated by leaving them in dry soil). A load cell was used to record the forces that earthworms produced as they moved through artificial burrows (holes that had been drilled through blocks of aluminium or Perspex). The radial pressure was calculated using the forces exerted and the dimensions of the artificial burrows. There was a negative correlation between burrow diameter and radial pressure, although radial pressure was independent of the length of the block through which the earthworms had burrowed. The highest radial pressures were produced by the anterior segments of the animal. Partial dehydration caused the earthworms to become quiescent, but did not decrease the radial pressure that the earthworms produced. It is suggested that coelomic fluid is retained in the anterior segments while the rest of the animal dehydrates. Dehydrated earthworms became lethargic, and we suggest that lethargy is due to the loss of coelomic fluid from the posterior segments. Coelomic fluid is known to be lost through dorsal pores. In burrowing species of earthworm such as Aporrectodea caliginosa, these pores are only present on the posterior segments.     

Toxicological effects of microcystin-LR on earthworm (<Emphasis Type="Italic">Eisenia fetida</Emphasis>) in soil

Microcystin-LR (MC-LR) is a cyclic heptapeptide toxin produced by cyanobacteria in eutrophic water. It can be transferred into soil–crop systems via irrigation and cyanobacterial paste fertilization. No studies have examined the potential toxicity of MC-LR to soil animals. Therefore, in the present study, the toxicological effects of MC-LR on earthworm (Eisenia fetida), including survival, growth, reproduction, oxidative stress, and cell viability, were investigated. The LC₅₀ of MC-LR was 0.149 μg cm^?2 at 72 h based on a filter paper test and 0.460 mg kg^?1 at 14 days based on an acute soil test. MC-LR seriously affected the reproduction of earthworms. Based on hatchability, the EC₅₀ of MC-LR was 0.268 mg kg^?1, similar to environmentally relevant concentrations of microcystins. The changes in activities of superoxide dismutase, guaiacol peroxidase, catalase, and glutathione peroxidase, together with the levels of glutathione and malondialdehyde, indicated that oxidative damage and lipid peroxidation played significant roles in MC-LR toxicity. In addition, the toxicity of MC-LR in earthworms increased despite degradation of MC-LR in soil over time, possibly due to the formation of toxic metabolites of MC-LR or the bioaccumulation of MC-LR in earthworms. A reduction in the neutral red retention time along with an increase in coelomocyte apoptosis with increasing MC-LR concentrations indicated a severe damage to viability. These results suggest that environmentally relevant MC-LR concentrations in agricultural soil may cause reproductive, biochemical, and cellular toxicity to Eisenia fetida. This information can be used in ecological risk assessments on MC-LR in soil.     

Transformation of nitrogen compounds and dynamics of microbial biomass in fresh casts of <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis>

A comparative assessment of the contents of total nitrogen, ammonium, nitrates, and fungal and bacterial biomasses and of the activity of nitrogen transformation in fresh casts of Aporrectodea caliginosa and in a soddy-podzolic soil was performed. The total content of nitrogen in the casts was similar to or slightly higher than that in the soil; the content of inorganic forms of nitrogen in the casts was significantly higher than in the soil. The intensities of ammonification, nitrification, and denitrification and the pool of microbial biomass with a predominance of fungi during the 1.5-week-long incubation were also significantly higher in the casts. The activity of nitrogen fixation in the casts was lower than that in the soil. In the course of the incubation, the values of these parameters in the casts became closer to those in the soil. The inhibition of fungi in the casts with cycloheximide resulted in an increasing content of inorganic nitrogen and a higher activity of denitrification. In our opinion, this phenomenon is related to the fact that limitation of the fungal growth decreases the intensity of immobilization of nitrates and the fungal competition with denitrifying bacteria for available carbon. It was supposed that the activation of the fungal growth via application of plant substrates into the soil with a high population density of earthworms could suppress the emission of gaseous nitrogen compounds. The soil processing by earthworms had a positive effect on the soil properties.     

Dissipation of fomesafen in biochar-amended soil and its availability to corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) and earthworm (<Emphasis Type="Italic">Eisenia fetida</Emphasis>)

Purpose

Biochar application has been shown to be effective in improving soil fertility and sequestering soil contaminants. However, the impact of biochar amendments on the environmental fate of pesticides and the bioavailability of pesticides to living organisms in the soil environment is still not fully understood.

Materials and methods

Dissipation of fomesafen and its bioavailability to corn (Zea mays L.) and the earthworm Eisenia fetida in an agricultural soil amended with three different rates of rice hull biochar (0.5, 1, and 2 % (w/w)) under laboratory conditions was investigated.

Results and discussion

Biochar amendment significantly increased the DT₅₀ of fomesafen from 34 days in unamended soil to 160 days in 2 % biochar-amended soil. Furthermore, biochar amendment decreased fomesafen concentration in soil pore water resulting in lower plant uptake of the pesticide. In this case, total plant residue and soil pore water concentrations of fomesafen in 2 % biochar-amended soil decreased to 0.29 % and 0.28–45 % of that in the control, respectively. Similar results were obtained for bioavailability of fomesafen in earthworms, as the earthworm residue and soil pore water concentration of fomesafen in 2 % biochar-amended soil declined to 0.38–45 and 0.47–0.50 % compared to the level of the control, respectively.

Conclusions

As biochar could markedly reduce the concentration of fomesafen in soil pore water and subsequently reduce plant and earthworm uptake of fomesafen from contaminated soil, biochar amendment could be considered an appropriate option for immobilizing fomesafen in soils, protecting nontarget organisms from fomesafen contamination.

     

Production and cytogenetics of trigeneric hybrid involving <Emphasis Type="Italic">Triticum</Emphasis>, <Emphasis Type="Italic">Psathyrostachys</Emphasis> and <Emphasis Type="Italic">Secale</Emphasis>

Trigeneric hybrids may help establish evolutionary relationships among different genomes present in the same cellular-genetic background, and also offers the possibility to transfer different alien characters into cultivated wheat. In this study, a new trigeneric hybrid involving species from the Triticum, Psathyrostachys and Secale was synthesized by crossing wheat-P. huashanica amphiploid (PHW-SA) with wheat-S. cereale amphiploid (Zhongsi 828). The crossability of F₁ hybrid was high with 35.13%, and the fertility was 41.95%. The morphological characteristics of F₁ plants resembled the parent Zhongsi 828. The trigeneric hybrids pollen mother cells (PMCs) regularly revealed averagely 19.88 univalents, 9.63 ring bivalents, 3.97 rod bivalents, 0.60 trivalents and 0.03 tetravalents per cell. Multivalents consisted of trivalents and tetravalents can be observed in 52.7% of cells. A variation of abnormal lagging chromosome, micronuclei and chromosome bridge were formed at anaphase I and telophase II. The mean chromosomes number of F₂ progenies was 2n = 46.13, and the distribution range was 42–53. GISH results revealed that most F₂ plants had 6–12 S. cereale chromosomes, and only 0–2 P. huashanica chromosomes were detected. The results indicated that S. cereale chromosomes can be preferentially transmitted in the F₂ progenies of trigeneric hybrid than P. huashanica chromosomes. A survey of disease resistances revealed that the stripe rust resistance from the PHW-SA were completely expressed in the F₁ and some F₂ plants. The trigeneric hybrid could be a useful bridge for the transference of P. huashanica and S. cereale chromatins to common wheat.     

Putative Role of <Emphasis Type="Italic">Flavobacterium</Emphasis>, <Emphasis Type="Italic">Dokdonella</Emphasis> and <Emphasis Type="Italic">Methylophilus</Emphasis> Strains in Paracetamol Biodegradation

Paracetamol, the most widely and globally used analgesic and antipyretic, is easily accumulated in aquatic environments. In the present study, the biodegradation of paracetamol in different media (one for general growth, one specific for sulfate reducing bacteria, a mineral salts medium and municipal wastewater) inoculated with two types of sludge (from anaerobic lagoon and from oxidation ditch) under different oxygenic conditions (anoxic; moderate oxygenation in open flasks and high oxygenation by aeration) was investigated. In addition, bacteria with relative abundances increasing simultaneously with paracetamol degradation, when this drug was the only carbon source, thus with a putative role in its degradation, were identified using 16S rRNA gene sequences. The results show that aerobic microorganisms had a major role in the degradation of paracetamol, with 50 mg/L totally removed from municipal wastewater after 2 days incubation with aeration, and that the metabolites 4-aminophenol and hydroquinone plus one compound not identified in this work were produced in the process. The identification of bacteria with a role in the degradation of paracetamol revealed a strain from genus Pseudomonas with the highest final relative abundance of 21.2%, confirming previous works reporting strains of this genus as paracetamol decomposers. Besides, genera Flavobacterium, Dokdonella and Methylophilus were also in evidence, with initial relative abundances of 1.66%, 1.48 and 0.00% (not detected) in the inoculum and 6.91%, 3.80 and 3.83% after incubation, respectively. Therefore, a putative role of these genera in paracetamol biodegradation is suggested for the first time.

Open image in new window

Graphical Abstract ?

     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

<Emphasis Type="Italic">Trichoderma</Emphasis> improves the growth of <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Bio-fertilizer application has been proposed as a strategy for enhancing soil fertility, regulating soil microflora composition, and improving crop yields, and it has been widely applied in the agricultural yields. However, the application of bio-fertilizer in grassland has been poorly studied. We conducted in situ and pot experiments to investigate the practical effects of different fertilization regimes on Leymus chinensis growth, with a focus on the potential microecological mechanisms underlying the responses of soil microbial composition. L. chinensis biomass was significantly (P?<?0.05) increased by treatment with 6000 kg ha^?1 of Trichoderma bio-fertilizer compared with other treatments. We found a positive (R² =?0.6274, P <?0.001) correlation between bacterial alpha diversity and L. chinensis biomass. Hierarchical cluster analysis and nonmetric multidimensional scaling (NMDS) revealed that soil bacterial and fungal community compositions were all separated according to the fertilization regime used. The relative abundance of the most beneficial genera in bio-fertilizer (BOF) (6000 kg ha^?1Trichoderma bio-fertilizer) was significantly higher than in organic fertilizer (OF) (6000 kg ha^?1 organic fertilizer) or in CK (non-amend fertilizer), there the potential pathogenic genera were reduced. There were significant negative (P?<?0.05) correlations between L. chinensis biomass and the relative abundance of several potential pathogenic genera. However, the relative abundance of most beneficial genera were significantly (P?<?0.05) positively correlated with L. chinensis biomass. Soil properties had different effects on these beneficial and on these pathogenic genera, further influencing L. chinensis biomass.     

<Emphasis Type="Italic">Achromobacter insolitus</Emphasis> and <Emphasis Type="Italic">Zoogloea ramigera</Emphasis> associated with wheat plants (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

This study reports for the first time the presence of diazotrophic bacteria belonging to the genera Achromobacter and Zoogloea associated with wheat plants. These bacterial strains were identified by the analysis of 16S rDNA sequences. The bacterium IAC-AT-8 was identified as Azospirillum brasiliense, whereas isolates IAC-HT-11 and IAC-HT-12 were identified as Achromobacter insolitus and Zoogloea ramigera, respectively. A greenhouse experiment involving a non-sterilized soil was carried out with the aim to study the endophytic feature of these strains. After 40 days from inoculation, all the strains were in the inner of roots, but they were not detected in soil. In order to assess the location inside wheat plants, an experiment was conducted under axenic conditions. Fifteen days after inoculation, preparations of inoculated plants were observed by the scanning electron microscope, using the cryofracture technique, and by the transmission electron microscope. It was observed that all isolates were present on the external part of the roots and in the inner part at the elongation region, in cortex cells, but not in the endodermis or in the vascular bundle region. No colonizing bacterial cells were observed in wheat leaves.     

Spatial variation in soil compaction,and the burrowing activity of the earthworm<Emphasis Type="Italic"> Aporrectodea caliginosa</Emphasis>

The effects of soil compaction on earthworm ( Aporrectodea caliginosa nocturna) activity were studied using pot experiments. Two compaction pressures were used when packing the pots; loose soil was packed by applying a pressure of 96 kPa, and compact soil was packed using a compaction pressure of 386 kPa. Split pots which contained both loose and compact soil were also used. In split pots peripheral burrows (those next to the pot wall), were generally longer in the loose half of the pot than they were in the compact half. This implies that earthworms preferred the loose soil. An important finding was that peripheral burrows in the split pots were longer than those made in pots packed uniformly with either loose or compact soil. Our data suggest that the increase in activity in split pots is a response to spatial variation in soil compaction. In the split pots the earthworms tended to avoid the compact half, and we discuss how the avoidance response in the split pots may have increased the proportion of burrows in these pots that were peripheral.     

Salt tolerance in <Emphasis Type="Italic">Zea mays</Emphasis> (L). following inoculation with <Emphasis Type="Italic">Rhizobium</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis>

This study aimed to investigate the effect of inoculation with plant growth-promoting Rhizobium and Pseudomonas species on NaCl-affected maize. Two cultivars of maize (cv. Agaiti 2002 and cv. Av 4001) selected on the basis of their yield potential were grown in pots outdoors under natural conditions during July. Microorganisms were applied at seedling stage and salt stress was induced 21 days after sowing and maintained up to 50% flowering after 120 days of stress. The salt treatment caused a detrimental effect on growth and development of plants. Co-inoculation resulted in some positive adaptative responses of maize plants under salinity. The salt tolerance from inoculation was generally mediated by decreases in electrolyte leakage and in osmotic potential, an increase in osmoregulant (proline) production, maintenance of relative water content of leaves, and selective uptake of K ions. Generally, the microbial strain acted synergistically. However, under unstressed conditions, Rhizobium was more effective than Pseudomonas but under salt stress the favorable effect was observed even if some exceptions were also observed. The maize cv. Agaiti 2002 appeared to be more responsive to inoculation and was relatively less tolerant to salt compared to that of cv. Av 4001.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

The role of earthworms (<Emphasis Type="Italic">Eisenia fetida</Emphasis>) in influencing bioavailability of heavy metals in soils

The effects of earthworm (Eisenia fetida) activity on soil pH, dissolved organic carbon (DOC), microbial populations, fraction distribution and bioavailability of heavy metals (Zn, Cu, Cr, Cd, Co, Ni, and Pb) in five Chinese soils were investigated using pot experiments. A three-step extraction procedure recommended by the European Community Bureau of Reference (BCR; now Standards, Measurements and Testing Programme of the European Community) was used to fractionate the metals in soils into water soluble, exchangeable and carbonate bound (B1), Fe-oxides and Mn-oxides bound (B2) and organic matter and sulfide bound (B3). After the soils were treated with earthworms, the soil pH, water-soluble metal fraction and DOC increased. A significant correlation was obtained between the increased DOC and the increased metals in the water-soluble fraction. The heavy metals in fraction B1 increased after earthworm treatments, while those in fraction B3 decreased. No significant differences were observed for heavy metals in fraction B2. The microbial populations in soil were enumerated with the dilution plate method using several media in the presence of earthworms. The microbial populations increased due to earthworm activity. The biomass of wheat shoots and roots, and the heavy metal concentrations in wheat roots and shoots, were also increased due to the earthworm activity. The present results demonstrated that earthworm activity increases the mobility and bioavailability of heavy metals in soils.     

Characterization of fertile amphidiploid between <Emphasis Type="Italic">Raphanus sativus</Emphasis> and <Emphasis Type="Italic">Brassica alboglabra</Emphasis> and the crossability with <Emphasis Type="Italic">Brassica</Emphasis> species

A fertile amphidiploid × Brassicoraphanus (RRCC, 2n = 36) between Raphanus sativus cv. HQ-04 (2n = 18, RR) and Brassica alboglabra Bailey (2n = 18, CC) was synthesized and successive selections for seed fertility were made from F₄ to F₁₀. F₁₀ plants exhibited good fertility with 14.9 seeds per siliqua and 32.3 g seeds per plant. Cytological observation revealed that frequent secondary pairing occurred among 3 chromosome pairs in pollen mother cells of plants (F₄) with lower fertility, but not of plants with high fertility (F₁₀). GISH analysis indicated that these F₁₀ plants included the expected 18 chromosomes from R. sativus and B. alboglabra, respectively, but they lost approximately 27.6% R. sativus and 35.6% B. alboglabra AFLP (amplified fragment length polymorphism) bands. The crossability of the Raphanobrassica with R. sativus and 5 Brassica species (13 cultivars) were investigated. Seeds or F₁ seedlings were easy to be produced from crosses × Brassicoraphanus × R. sativus, and B. napus, B. juncea and B. carinata × Brassicoraphanus. Fewer seeds or seedlings were obtained from crosses × Brassicoraphanus × B. napus, B. juncea and B. carinata. However, few seeds were harvested in the reciprocals of × Brassicoraphanus with B. rapa and B. oleracea. The possible cause of fertility improvements and the potential of the present × Brassicoraphanus for breeding were discussed.     

Phylogenetic relationships between <Emphasis Type="Italic">Ensete</Emphasis> and <Emphasis Type="Italic">Musa</Emphasis> species as revealed by the <Emphasis Type="Italic">trnT trnF</Emphasis> region of cpDNA

Complete sequences of transcribed spacers and introns from the trnT trnF region of chloroplast DNA (cp DNA) were generated from Musaceae species to establish the phylogenetic relationships among 3 species of Ensete including the economically important Ensete ventricosum (Welw.) Cheesman and 13 species of Musa. Parsimony analysis and pair wise distance data produced a single tree, with Ensete and Musa as clearly distinguished clades. Six Musa and three Ensete clades were generated. The topology of these clades did not change when the data were split into spacers and introns, although the split resulted in poor bootstrap support. Removing a hotspot from the entire data set improved clade support. The clades produced are discussed with reference to existing taxonomic and phylogenetic treatments. In contrast to previous suggestions, most of the Rhodochlamys species that we investigated clustered together with strong support establishing their distinctiveness from the Musa species studied. Ensete glaucum (Roxb.) Cheesman and Musa beccartii Simmonds appear to represent ancestral forms of Ensete and Musa, respectively for the presently studied species, and both genera have a common ancestor that is yet to be established. Our data also show that E. ventricosum cannot be reduced to E. glaucum, nor can E. gilleti (De Wild.) Cheesman be reduced to E. ventricosum, as some authorities have suggested. Ensete gilleti or a species very close to it appears to be the ancestral species of E. ventricosum.     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

A new mycorrhizal helper bacterium, <Emphasis Type="Italic">Ralstonia</Emphasis> species,in the ectomycorrhizal symbiosis between <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">Suillus granulatus</Emphasis>

In a Robinia-pseudoacacia-dominated coastal forest in Tottori prefecture Japan, the growth and survival of Pinus thunbergii seedlings and the natural regeneration of P. thunbergii was disturbed by R. pseudoacacia. In order to improve the growth of P. thunbergii seedling in the Tottori sand dune, we tried to find a mycorrhiza helper bacteria (MHB) from P. thunbergii mycorrhizosphere in a Tottori sand dune. Two MHB, Ralstonia sp. and Bacillus subtilis, were selected from the nine bacterial species isolated from the mycorrhizosphere of P. thunbergii. The bacterial effect on the ectomycorrhizal fungus Suillus granulatus was investigated by confrontation assay and a microcosm experiment. The confrontation assay showed that Ralstonia sp. promoted the hyphal growth of S. granulatus. Moreover, the S. granulatus–P. thunbergii symbiosis was significantly stimulated by Ralstonia sp. and B. subtilis. Ralstonia sp. and B. subtilis were regarded as MHB associated with P. thunbergii. This is the first report of Ralstonia sp. as an MHB.     

Effects of Single,Binary and Quinary Mixtures of Phenanthrene and Its N-PAHs on <Emphasis Type="Italic">Eisenia fetida</Emphasis> in Soil

It is now acknowledged that aromatic hydrocarbons present in contaminated soils occur in mixtures. The effect of single, binary and quinary mixtures of phenanthrene and selected nitrogen-containing polycyclic aromatic hydrocarbons (N-PAHs) were investigated on the survival, growth and behavioural index of earthworms (Eisenia fetida) over a 21-day incubation in soil. The results showed that the LC₅₀ values ranged from (not detected) ND–329.3 mg kg^?1 (single mixture), ND–219.8 mg kg^?1 (binary mixtures) to 148.4 mg kg^?1 (quinary mixture), while the EC₅₀ values (based on weight loss) ranged from 13.3–148.4 mg kg^?1 (single mixture), 63.8–148.4 mg kg^?1 (binary mixture) to 24.2 mg kg^?1 (quinary mixture). Greater impacts were recorded where N-PAHs are present with phenanthrene. Further, behavioural index of E. fetida was affected after 24-h exposure to N-PAH-amended soils. Among the N-PAHs however, benzo[h]quinoline recorded the greatest impact on the survival, growth and behavioural index of E. fetida in soil. Findings from this study showed that three ring-N-PAHs are more toxic than phenanthrene as expected from their physico-chemical properties. The binary and quinary mixtures of phenanthrene and N-PAHs in soil intensified toxicity, suggesting that PAHs-N-PAHs mixtures represent greater risk to soil biota.     

Distribution and fractionation of cadmium in soil aggregates affected by earthworms (<Emphasis Type="Italic">Eisenia fetida</Emphasis>) and manure compost

Purpose

The objectives of this study were (1) to investigate the effects of manure compost and earthworms on Cd mobility in Cd-contaminated soil, (2) to test whether the bioturbation of earthworms reduces the immobilization effect of the manure compost when they are combined, and (3) to explore the distribution of Cd in aggregates formed by earthworms with corresponding fractionation analysis.

Materials and methods

A laboratory experiment was conducted to evaluate the effect of either or both application of manure compost and the earthworms Eisenia fetida into cadmium historically contaminated soil on cadmium mobility. Soil characteristics and metal concentrations in earthworms and soil were measured, and soil aggregates in the mesocosms were separated for Cd fraction analysis based on four steps sequential extraction.

Results and discussion

Manure compost reduced mobile Cd based on CaCl₂ extraction and Toxicity Characteristic Leaching Procedure (TCLP) test by 60–95 and 25–30 %, respectively. However, earthworm application alone increased Cd mobility by 9–15 %. Besides, in the presence of manure compost, earthworms further immobilized cadmium to a slight extent. The interaction effect of manure compost and earthworms combined on Cd immobilization suggested that earthworms promoted the formation of large macroaggregates (>2 mm) and the redistribution of Cd concentration in soil aggregates. Additionally, earthworms reduced carbonate fraction of Cd from 42.3–49.6 to 6.3–19.5 % in different aggregates, respectively. And, residual fraction of Cd increased from 33.9–42.2 to 63.9–77.5 % simultaneously. The results may be due to the thorough mixture of phosphates and organic matter with cadmium during bioturbation on account of the available form of phosphorus, nitrogen, and cadmium changing to the more recalcitrant form.

Conclusions

Manure compost addition increased the soil pH, phosphorus, nitrogen, and organic carbon content, and decreased Cd mobility. The application of earthworms and manure compost combined exhibited higher efficiency for cadmium immobilization, which can be used for Cd remediation due to the redistribution of Cd concentration in soil aggregates and the transformation of soluble Cd to the residual precipitate fraction.