•The phylogenetic relationships of Amynthas species and species-groups from South China are addressed.•Two separate groups, each containing a diversity of spermathecal pore configurations and each corresponding to one of the main collection areas, were revealed in the phylogeny.•Sims & Easton’s species groups are non-monophyletic except a parthenogenetic group.Our knowledge of the phylogeny of the earthworm genus Amynthas under the family Megascolecidae, which is comprised of a huge number of species, is very limited compared to the better-known and much smaller family Lumbricidae. In order to investigate the phylogenetic relationships among the species within the genus Amynthas, which is the largest genus of the Megascolecidae family, nuclear and mitochondrial DNA sequences of 77 species, including 76 in-group Amynthas species collected from South China and 1 out-group species, were analyzed. A 5402 bp segments composed of whole nuclear 18 S rDNA and the mitochondrial genes COI, COII, ND1, 12 S, and 16 S was assembled from 77 species. Maximum Likelihood and Bayesian analyses of the concatenated sequences were performed. The results revealed evolution of two geographically independent lineages, both showing the ancestral state of two pairs of spermatheca (Sp.p 7/8/9). We found the species groups described by Sims and Easton (1972) to be non-monophyletic, and the origin of the parthenogenetic species group to likely be a quadthecal ancestor. These results provide modest evidence in support of an Indochinese peninsula origin of the Chinese Amynthas species and divergence of the genus once it had spread to mainland China. The findings of this study are consistent with a divergence scenario that resulted in at least one branch spreading to the Southeast of China and another branch spreading to the Southwest of China, but further research is required to confirm this interpretation of the Amynthas phylogeny.Download high-res image (165KB)Download full-size image

•We isolate a bacteria with antagonist activity against R. Solanacearum.•The strain VIH2 was identified as Pseudomonas aeruginosa.•Our data supported the idea that HSI-I T6SS plays a crucial role in the antagonistic action of strain VIH2 against R. solanacearum.The aim of this study was to isolates with antagonist activity against R. solanacearum. Thirty-two bacterial isolates were obtained from samples, and they were screened for potential antagonistic activity against R. Solanacearum. Using the agar spot method, ten out of the 21 tested bacteria showed antilisterial activity. VIH2 had the highest inhibitory effect on the growth of R. Solanacearum. Based on 16S rDNA and Biolog test analysis, the strain VIH2 was identified as Pseudomonas aeruginosa. Single-factor and Response Surface Methodology experiments were used to optimize the culture medium and conditions. This study was to explore whether the hemolysin-co-regulated protein secretion island I (HSI-I)-encoded type VI secretion system (T6SS) in Pseudomonas can be used as a biological control approach against Ralstonia solanacearum under field conditions. Bacterial competition assay showed that the HSI-I type T6SS of strain VIH2 exhibited dramatic antibacterial killing activity against R. solanacearum. The HSI-I T6SS of P. aeruginosa was regulated by the ppKA gene. We disrupted the gene ppKA in VIH2 by a single crossover to yield the VIH2 (ΔppKA) mutant. The antagonism of VIH2 was significantly decreased by ppKA gene disruption. In conclusion, our data supported the idea that HSI-I T6SS plays a crucial role in the antagonistic action of strain VIH2 against R. solanacearum. This alternative approach for antagonism against R. solanacearum might help develop attenuated strains of engineered bacteria for biological control.

•Soil iron and aluminum minerals showed great affinity for the laccase.•Adsorbed laccases retained 26–64% of the activity of the free enzyme, and showed increased Km values and decreased Vmax values versus the free laccase.•Mineral adsorption enhanced catalytic activity of laccase under extra acid conditions and decreased the thermal stability.•Mineral adsorption increased Ea and ΔHa values of laccase.•Mineral adsorption improved the resistance to proteolysis and extended the lifespan of laccase.Laccases play an important role in the degradation of soil phenol or phenol-like substance and can be potentially used in soil remediation through immobilization. Iron and aluminum minerals can adsorb extracellular enzymes in soil environment. In the present study, we investigated the adsorptive interaction of laccase, from the white-rot fungus Trametes versicolor, with soil iron and aluminum minerals and characterized the properties of the enzyme after adsorption to minerals. Results showed that both soil iron and aluminum minerals adsorbed great amount of laccase, independent of the mineral specific surface areas. Adsorbed laccases retained 26–64% of the activity of the free enzyme. Compared to the free laccase, all adsorbed laccases showed higher Km values and lower Vmax values, indicating a reduced enzyme-substrate affinity and a lower rate of substrate conversion in reactions catalyzed by the adsorbed laccase. Adsorbed laccases exhibited increased catalytic activities compared to the free laccase at low pH, implying the suitable application of iron and aluminum mineral-adsorbed T. versicolor laccase in soil bioremediation, especially in acid soils. In terms of the thermal profiles, adsorbed laccases showed decreased thermal stability and higher temperature sensitivity relative to the free laccase. Moreover, adsorption improved the resistance of laccase to proteolysis and extended the lifespan of laccase. Our results implied that adsorbed T. versicolor laccase on soil iron and aluminum minerals had promising potential in soil remediation.

An experiment was conducted to investigate the effects of earthworm (Metaphire guillelmi) activities on rice photosynthates distribution in plant–soil system through 14C pulse-labelling method. Rice was planted in pots, and maize straw was mulched on the surface with or without earthworms. Rice plants at tillering stage or heading stage were labelled with 14CO2. Plant and soil were sampled 15 days after labelling at the tillering or heading stage and at harvest. Rice growth was inhibited by earthworms (M. guillelmi) at early stage, but the inhibition disappeared at later stage. Earthworms significantly (P < 0.01) increased the 14C percentage in root at day 15 after tillering stage labelling, but the effect disappeared at harvest. Earthworms (M. guillelmi) significantly (P < 0.01) increased the 14C percentage in root at day 15 after heading stage labelling and increased 14C percentage in soil at harvest. Earthworms decreased the percentages of total organic 14C (TO14C) present as microbial biomass 14C (MB14C) and increased the percentages of total organic 14C present as dissolved organic 14C (DO14C) at all sampling times. It is suggested that earthworms might alter the transfer of plant photosynthates from the aboveground to the belowground, and thus, soil active C pool. However, these data should be also confirmed in the field.

The effects of IAA-producing bacteria (Pseudomonas sp. and Burkholderia sp.) and bacterial-feeding nematodes (Cephalobus sp.) on Arabidopsis thaliana (Ecotype, Columbia) root growth were investigated in pots with natural soil under controlled conditions. The soil, nematodes and root morphology of Arabidopsis were analysed at regular intervals. In natural soil, the bacterial-feeding nematodes grazing on bacteria stimulated both the microbial growth and activity and increased the mineral nitrogen and IAA contents. The analysis of the root architecture (total length, the number of tips, average diameter and surface area) of the A. thaliana seedlings revealed a highly branched root system, with longer and thinner roots in the presence of the IAA-producing bacteria and bacterial-feeding nematodes. On the basis of the above findings, it was concluded that the IAA-producing bacteria and bacterial-feeding nematodes improved the root growth of the A. thaliana seedlings in natural soil. The validity of our conclusion may be extended to future applications in agriculture.Highlights► The addition of bacteria and nematodes stimulated microbial activity. ► The addition of bacteria and nematodes increased mineral nitrogen and IAA content. ► The addition of nematodes resulted in longer and thinner Arabidopsis roots. ► The promotion of Arabidopsis root growth was better for nematodes than bacteria. ► We suggest adding a combination of nematodes and bacteria to promote plant growth.

•Co-polluted soil with antibiotics and nitrogen has become a potential threat.•Antibiotic presence significantly inhibited the denitrification process in soil.•Enforced denitrification prompted the decline in antibiotic and ARG levels.•The higher the DNG level, the more dramatic was the progression of denitrification.•Clearly negative correlation was detected between DNG and ARG abundance.Mixed contamination of nitrate and antibiotics/antibiotic-resistant genes (ARGs) is an emerging environmental risk to farmland soil. This is the first study to explore the role of excessive anthropogenic nitrate input in the anoxic dissipation of soil antibiotic/ARGs. During the initial 10 days of incubation, the presence of soil antibiotics significantly inhibited NO3− dissipation, N2O production rate, and denitrifying genes (DNGs) abundance in soil (p < 0.05). Between days 10 and 30, by contrast, enhanced denitrification clearly prompted the decline in antibiotic contents and ARG abundance. Significantly negative correlations were detected between DNGs and ARGs, suggesting that the higher the DNG activity, the more dramatic is the denitrification and the greater are the antibiotic dissipation and ARG abundance. This study provides crucial knowledge for understanding the mutual interaction between soil DNGs and ARGs in the enhanced anoxic denitrification condition.Download high-res image (195KB)Download full-size image

A long-term field experiment (started at 2003) was conducted to determine the effects of different rice cultivation methods on growth characteristics and grain yield of late-season rice under double-rice cropping system in seasonal drought region of southeast China (Yujiang County, Jiangxi Province). The rice cultivation methods included no-tillage and flooded rice cultivation (N-F), no-tillage and non-flooded rice cultivation with straw mulching (N-SM), and no-tillage and non-flooded rice cultivation without straw mulching (N-ZM). There was no significant difference in rice grain yield between the N-SM and N-F treatments. However, the rice grain yields in the N-SM and N-F treatments were significantly higher than that in the N-ZM treatment. The late-season rice plants in the N-SM treatment had significantly higher numbers of effective panicles and total grains per hill compared with those in the N-ZM treatment. The above-ground dry matter of late-season rice was similar between the N-SM and N-F treatments. Compared with the N-F treatment, the N-ZM and N-SM treatments significantly decreased the leaf area at the heading stage. Moreover, the N-SM treatment could significantly increase total root length and root tip number at the grain-filling stage compared with the N-ZM treatment.

The distribution of heavy metals in earthworms has been widely studied, highlighting the importance of the fate of these metals. However, little information is available on the distribution of hydrophobic organic contaminants (HOCs) within earthworms. The aim of this study was to propose a hierarchic method to study the distribution of phenanthrene (PHE), a typical HOC, in Eisenia fetida at several levels: sub-organism (pre-clitellum, clitellum and post-clitellum), tissue (body wall, gut and body fluid) and subcellular (intracellular and extracellular fractions). Earthworms were incubated in the soils amended with low (LC, 10 mg kg−1) and high concentrations (HC, 50 mg kg−1) of PHE and sampled at different time intervals. At the sub-organism level, the distribution of PHE was homogeneous among the sub-organism fractions in the LC treatment but heterogeneous in the HC treatment and gradually reached the following form of post-clitellum ≈ clitellum > pre-clitellum. The uptake and elimination kinetics of PHE in the sub-organism were well described by a one-compartment model. At the tissue level, the concentration of PHE followed the order of gut > body fluid > body wall; while at the subcellular level, the concentration of PHE in the extracellular fraction was 1.23 to 4.68 times higher than that in the intracellular fraction. Therefore, the simple circulatory system of earthworms may account for the PHE distribution at the sub-organism level. Partition pathways (passive diffusion) of PHE between the body wall, body fluid and gut as well as the processes of PHE entrance into the inner cellular compartment via passive diffusion were experimentally supported.

Earthworms can promote the bioremediation of contaminated soils through enhancing plant growth and microorganism development. The individual and combined effects of earthworms and ryegrass (Lolium multiflorum Lam.) on the removal of fluoranthene from a sandy-loam alluvial soil were investigated in a 70-d microcosm experiment. The experiment was set up in a complete factorial design with treatments in four replicates: without earthworms or ryegrass (control, CK), with earthworms only (E), with ryegrass only (P), and with both earthworms and ryegrass (EP). The residual fluoranthene, microbial biomass C, and polyphenol oxidase activity in the soil changed significantly (P < 0.01) with time. In general, the residual concentration of fluoranthene in the soil decreased sharply from 71.8–88.7 to 31.7–37.4 mg kg−1 in 14 d, and then decreased gradually to 19.7–30.5 mg kg−1 on the 70th d. The fluoranthene concentration left in the soil was the least with both earthworms and ryegrass, compared to the other treatments at the end of the experiment. Half-life times of fluoranthene in the E, P, and EP treatments were 17.8%-36.3% smaller than that of CK. More fluoranthene was absorbed by earthworms than ryegrass. However, the total amounts of fluoranthene accumulated in both the ryegrass and earthworms were small, only accounting for 0.01%-1.20% of the lost fluoranthene. Therefore, we assumed that microbial degradation would play a dominant functional role in fluoranthene removal from soil. We found that earthworms significantly increased microbial biomass C and polyphenol oxidase activity (P < 0.01) in the presence of ryegrass at the end of the experiment. Furthermore, microbial biomass C and polyphenol oxidase activity were significantly (P < 0.05) and negatively related to the residual fluoranthene concentration. This implied that earthworms might promote the removal of fluoranthene from soil via stimulating microbial biomass C and polyphenol oxidase activity.