Use of four microbial tests to assess the ecotoxicological hazard of contaminated sediments

Three single-species bioassays (Microtox, Selenastrum capricornutum, and Panagrellus redivivus) and a test using microbial communities developed on artificial substrates were used in a series of in situ and laboratory tests evaluating the ecotoxicological hazard of contaminated sediments at two sites on Lake Michigan: Waukegan (Illinois) Harbor and The Chicago Area Confined Disposal Facility Study. In the single-species tests, exposure to elutriates of contaminated sediments significantly inhibited bacterial luminescence, algal photosynthesis, and nematode survival and growth at polluted stations, while elutriates from control stations did not. The battery of three tests is a promising screening tool for in-place pollutants. Protozoan species richness and protozoan phototroph abundance were inhibited by elutriates from contaminated sites, but the abundance of heterotrophic protozoans was enhanced by sediment elutriates from some stations. Microbial community photosynthesis was significantly inhibited by most sediment elutriates, while community respiration was often stimulated; thus, functional responses paralleled the structural changes. Overall, the results of the microbial community tests were consistent with expected patterns of toxicity at the two sites on Lake Michigan. In general, single-species test results agreed with the community bioassays. Although community tests may be more realistic than single-species bioassays in predicting the impact of sediment contamination on actual ecosystems, caution must be exercised in interpreting the results.     

The effect of tetrahydrofuran on the enzymatic activity and microbial community in activated sludge from a sequencing batch reactor

Tetrahydrofuran (THF) is a toxic and carcinogenic compound that is commonly released from pharmaceutical, chemical and related industry wastewater. Currently, the effects of THF contamination on wastewater are unknown and a better understanding of THF toxicity toward biological processes in wastewater treatment is critical. In this study, we firstly investigated the toxic effects of THF on enzymatic activity and the microbial diversity in activated sludge from a sequencing batch reactor during long-term exposure to 10 mM THF. The activity of five enzymes (catalase, dehydrogenase, urease, phosphatase and protease) was remarkably decreased in the presence of 10 mM THF during a period of 85 days. Of these five affected enzymes, dehydrogenase activity was close to detection level limits and was nearly completely inhibited. Analysis of the microbial community demonstrated that THF, at a concentration of 10 mM, altered the distribution of microbes within the community and significantly decreased microbial diversity during long-term contamination, according to denaturing gradient gel electrophoresis (DGGE) analysis. The fraction of Actinobacteria increased in the community, while the fraction of Proteobacteria significantly decreased after THF exposure.     

Temporal bacterial diversity associated with metal-contaminated river sediments

The temporal activity, abundance and diversity of microbial communities were evaluated across a metal-contamination gradient around a Superfund site in Montana. In order to analyze short-term variability, samples were collected from six sites on four occasions over 12 months. Measurements of community activity, diversity and richness, quantified by dehydrogenase activity and through denaturant gradient gel electrophoresis (DGGE), respectively, were higher at contaminated sites adjacent to the smelter, relative to reference sites. 16S rRNA gene copy numbers, measured by quantitative PCR, showed seasonal variability, yet were generally higher within polluted sediments. Jaccard similarity coefficients of DGGE profiles, found sites to cluster based primarily on geographical proximity rather than geochemical similarities. Intra-site clustering of the most polluted sites also suggests a stable metal-tolerant community. Sequences from DGGE-extracted bands were predominantly Beta and Gammaproteobacteria, although the communities at all sites generally maintained a diverse phylogeny changing in composition throughout the sampling period. Spearman’s rank correlations analysis found statistically significant relationships between community composition and organic carbon (r-value = 0.786) and metals (r-values As = 0.65; Cu = 0.63; Zn = 0.62). A diverse and abundant community at the most polluted site indicates that historical contamination selects for a metal-resistant microbial community, a finding that must be accounted for when using the microbial community within ecosystem monitoring studies. This study highlights the importance of using multiple time-points to draw conclusions on the affect of metal contamination.     

Long term effect of methylparathion contamination on soil microbial community diversity estimated by 16S rRNA gene cloning

The long-term effects of methylparathion contamination on the diversity of soil microbial community was investigated by a culture-independent approach using small subunit ribosomal RNA (SSU rRNA) gene-based cloning. Microbial DNA extracted from both the control soil sample and methylparathion contaminated soil sample was subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences. From the PCR amplification product, clone libraries were constructed for both samples. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and HhaI. A total of 603 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, the phylotype richness, frequency distribution (evenness) of the two clone libraries were compared by using a variety of diversity indices. Phylogenetic analysis of the sequences of the dominant phylotypes revealed that the bacterial communities changed noticeably. In the control soil, the dominant bacterial groups included a member of a novel bacterial division, the bacillus genus, and a member of α-proteobacteria, while in methylparathion contaminated soil, the dominant phylotypes were replaced by a member of the flexibactera-cytophaga-bacteroides division and two members of the γ-proteobacteria subdivision. This is the first report of the long-term effects of methylparathion (one of the major pesticides widely used in developing countries) on soil microbial community diversity and structure by a culture-independent method, and provides the evidences to assess the long-term environmental toxicological effects of methylparathion from the microbial community viewpoint.     

Mangrove trees affect the community structure and distribution of anammox bacteria at an anthropogenic-polluted mangrove in the Pearl River Delta reflected by 16S rRNA and hydrazine oxidoreductase (HZO) encoding gene analyses

Anaerobic ammonium oxidizing (anammox) bacterial community structures were investigated in surface (1–2 cm) and lower (20–21 cm) layers of mangrove sediments at sites located immediately to the mangrove trees (S0), 10 m (S1) and 1000 m (S2) away from mangrove trees in a polluted area of the Pearl River Delta. At S0, both 16S rRNA and hydrazine oxidoreductase (HZO) encoding genes of anammox bacteria showed high diversity in lower layer sediments, but they were not detectable in lower layer sediments in mangrove forest. S1 and S2 shared similar anammox bacteria communities in both surface and lower layers, which were quite different from that of S0. At all three locations, higher richness of anammox bacteria was detected in the surface layer than the lower layer; 16S rRNA genes revealed anammox bacteria were composed by four phylogenetic clusters affiliated with the “Scalindua” genus, and one group related to the potential anammox bacteria; while the hzo genes showed that in addition to sequences related to the “Scalindua”, sequences affiliated with genera of “Kuenenia”, “Brocadia”, and “Jettenia” were also detected in mangrove sediments. Furthermore, hzo gene abundances decreased from 36.5 × 10⁴ to 11.0 × 10⁴ copies/gram dry sediment in lower layer sediments while increased from below detection limit to 31.5 × 10⁴ copies/gram dry sediment in lower layer sediments from S0 to S2. The results indicated that anammox bacteria communities might be strongly influenced by mangrove trees. In addition, the correlation analysis showed the redox potential and the molar ratio of ammonium to nitrite in sediments might be important factors affecting the diversity and distribution of anammox bacteria in mangrove sediments.     

Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China

Surface soils from Guiyu, China (an intense e-waste processing center) were analyzed for persistent organic pollutants (POPs) and variations in composition of the resident bacterial communities. Denaturing Gradient Gel Electrophoresis analysis of bacterial 16S rRNA gene showed that e-waste pollution altered the bacterial community structure by promoting changes in species composition and species richness. Bacterial diversity was not decreased at e-waste open-burning sites, compared with a non e-waste site (reservoir site), due to flourishing of possible POPs-consuming bacterial cohorts. PAH-incubated experiments confirmed that different levels of PAHs might affect the bacterial community by suppressing or favoring certain groups of bacteria, for instance, uncultured Clostridium sp. and Massilia sp., respectively. Taxonomic analysis indicated β-proteobacteria and Firmicutes were abundant bacterial lineages in PAH-polluted soils. This study is the first reporting bacterial community structures at e-waste processing sites, and indicated that crude processing of e-waste has become a biohazard to the terrestrial environment warranting more extensive studies of microbial communities at e-waste polluted environments.     

Changes in bacterial diversity and community structure following pesticides addition to soil estimated by cultivation technique

An experiment was conducted under laboratory conditions to investigate the effect of increasing concentrations of fenitrothion (2, 10 and 200 mg a.i./kg soil), diuron (1.5, 7.5 and 150 mg a.i./kg soil) and thiram (3.5, 17.5 and 350 mg a.i./kg soil) on soil respiration, bacterial counts and changes in culturable fraction of soil bacteria. To ascertain these changes, the community structure, bacterial biodiversity and process of colony formation, based on the r/K strategy concept, EP- and CD-indices and the FOR model, respectively, were determined. The results showed that the measured parameters were generally unaffected by the lowest dosages of pesticides, corresponding to the recommended field rates. The highest dosages of fenitrothion and thiram suppressed the peak SIR by 15–70% and 20–80%, respectively, while diuron increased respiration rate by 17–25% during the 28-day experiment. Also, the total numbers of bacteria increased in pesticide-treated soils. However, the reverse effect on day 1 and, in addition, in case of the highest dosages of insecticide on days 14 and 28, was observed. Analysis of the community structure revealed that in all soil treatments bacterial communities were generally dominated by K-strategists. Moreover, differences in the distribution of individual bacteria classes and the gradual domination of bacteria populations belonging to r-strategists during the experiment, as compared to control, was observed. However, on day 1, at the highest pesticide dosages, fast growing bacteria constituted only 1–10% of the total colonies number during 48 h of plate incubation, whereas in remaining samples they reached from 20 to 40% of total cfu. This effect, in case of fenitrothion, lasted till the end of the experiment. At the highest dosages of fenitrothion, diuron and at all dosages of thiram the decrease of biodiversity, as indicated by EP- and CD-indices on day 1, was found. At the next sampling time, no significant retarding or stimulating effect was detected. However, in case of CD values the higher differences were observed. The significant impact of pesticides on the physiological state of soil bacteria was not found. They were generally in dormant state (λ < 0.5), but immediately after pesticides application, the additional reduction of frequency of bacterial cell proliferation (max. decrease of λ value to 0.15 for thiram on day 14) and prolonged retardation time of colony appearance (max. increase of t _r value to 1.39 for fenitrothion on day 1) on agar plates were found.     

Combined effects of pyrene and UV-light on algae and bacteria in an arctic sediment

The phototoxicity potential of pyrene on natural algae and bacteria in an arctic sediment was evaluated and compared to that of pyrene treatment alone based on some functional and structural endpoints. Microcosms with arctic sediment from a shallow-water marine bay were incubated with pyrene under three different light regimes, natural sunlight with UV-light (Light_UV), natural sunlight without UV-light (Light) and dark. Presence of pyrene directly affected the algal community measured as decreased ¹⁴C-incorporation and decreased ammonium, nitrate and silicate uptake. These direct toxic effects from pyrene on the algae eventually led to indirect effects on the bacterial community observed as increased oxygen consumption. Besides the direct toxicity of pyrene to the benthic microbial community, indications of phototoxicity were found on the bacterial community detected as decreased oxygen consumption and increased bacterial diversity under Light_UV compared to Light. No indication of phototoxicity of pyrene was found on the algae, which might be due to the high direct toxicity of pyrene. Our results indicate that shallow arctic marine areas might be affected by phototoxicity if concentrations of oil components in the sediments increase.     

Effects of dispersed and adsorbed crude oil on microalgal and bacterial communities of cold seawater

Mesocosm facilities consisting of five 3.5 m³ stainless steel tanks filled with seawater from the St Lawrence Estuary (Québec, Canada) were used to conduct a 2 month experiment under the natural conditions prevailing at the end of the winter in subarctic environments, with seawater temperatures ranging from-1.5°C (surface ice cover) to 3°C. Various oil treatments were simulated in mesocosms: Forties crude oil was chemically dispersed, adsorbed onto an immersed substrate and spilled without any treatment. Total oil concentrations ranged from <1 mgl^-1 (untreated oil) to 44.6 mgl^-1 (dispersed oil). Contrasting with the parent crude oil and dispersed oil, the dissolved phase was enriched with low molecular weight polycyclic aromatic hydrocarbons (PAH). As revealed by pH variations, chlorophyll a contents and degraded pigments, the phytoplankton growth was inhibited early in tanks contaminated with dispersed and adsorbed oil. Although global measurements showed a recovery of the microalgal activity while the dispersed oil was diluted in a flow-through cascade system, the specific composition was quite different from the control, with an increase in small microflagellate species and a marked decline in the diversity of centric diatoms. Small microflagellates also dominated in the heavily oil-contaminated surface microlayer. The growth of viable heterotrophic bacteria (VHB) was immediately stimulated by both dispersed and adsorbed oil (10⁴–10⁵ colony forming units per ml) and oil-degrading bacteria (ODB) reached maximum densities (10² CFU ml^-1) later in the experiment. The adaptation of the indigenous community was assessed using the ODB/VHB ratio, which increased by ten times in the seawater contaminated with dispersed oil. No significant bacterial enhancement was observed in the tank that received untreated oil. No bacterial enrichment was found in the surface microlayer. In sediment traps, the bacterial density increased with the amount of total settling matter and oil residues.     

Investigation on bacterial community and diversity in the multilayer aquifer-aquitard system of the Pearl River Delta,China

Bacteria play an important role in groundwater chemistry. The groundwater resource in the Pearl River Delta (PRD) is responsible for 50 million people’s water requirement. High amount of ammonium, arsenic and methane had been reported in groundwater of the PRD, which was considered as the result of intensive bacterial metabolism in the multilayer aquifer-aquitard system. To investigate bacterial community in this system and its relation with groundwater chemistry, sediment and groundwater samples were taken from representative locations in the PRD at different lithological units. Bacterial 16S rRNA gene clone libraries were constructed for microbial identifications and community structures in different strata. Canonical correlation analysis between bacterial linages and environment variables (Cl^?, PO₄ ^3?, SO₄ ^2?, NH₄ ⁺) showed that community structures were significantly modified by geological conditions. Higher bacterial diversity was observed in samples from the Holocene aquitard M1 and aquifer T1, while in the older aquitard M2 and basal aquifer T2, bacterial diversity was much lower. Chloroflexi, γ-proteobacteria and δ-proteobacteria were the dominant phyla in the aquitard sediment. β-proteobacteria was the dominant phylum in sediment which was strongly influenced by fresh water. The results of this study demonstrated that bacterial community contains information of geological events such as sea transgression and deltaic evolution, and microbes in the aquitards have great potential in dominating groundwater quality in aquifers.     

Differential expression of two genes Oct‐4 and MUC5AC associates with poor outcome in patients with gastric cancer

Gastric cancer (GC) is the most frequent leading cause of cancer‐associated mortality worldwide that is linked to poor prognosis due to the lack of appropriate biomarkers. Our aim was to evaluate the MUC5AC and Oct‐4 expression levels in GC and to assess their association with clinical factors. Immunohistochemical analysis (IHC) and qRT‐PCR were performed in GC patients to examine the MUC5AC and Oct‐4 expression levels. The mRNA level of MUC5AC was significantly decreased in tumour tissues compared with non‐cancerous tissues (1.11 ± 0.69 vs 3.7 ± 0.71; P = .024). On the other hand, Oct‐4 mRNA level was upregulated in tumour tissues as compared to normal tissues (2. 86 ± 0.78 vs 0.87 ± 0.54; P = .0015). Decreased expression of MUC5AC was detected in 27 patients (67.5%), while high to moderate expression levels were observed in 13 cases (32.5%), but in normal tissues the expression levels of MUC5AC were increased (P = .001). The decreased expression of MUC5AC was associated with aggressive tumour characteristics, such as TNM stage (P = .023), histologic type (P = .012) and lymph node metastasis (P = .001). High expression of Oct‐4 was detected in 24 tumour tissues (60%), while 16 cases (40%) showed low expression level. Increased Oct‐4 expression was correlated with clinicopathological characteristics such TNM stage (P = .002), histologic type (P = .008) and lymph node metastasis (P = .001). Our results showed that high Oct‐4 expression and the reduction of MUC5AC expression may be involved in the progression and an unfavorable prognosis of GC.     

Impaired megabenthic community structure caused by summer hypoxia in a eutrophic coastal bay

Eutrophication and hypoxia are major problems affecting the health of coastal ecosystems throughout the world. Tokyo Bay, Japan, is a eutrophic coastal area where the abundance of the megabenthic community has been decreasing. To assess factors associated with the impaired biota, seasonal surveys of the megabenthic community and water and sediment quality were conducted in the bay. Cluster analysis showed a difference in the community structure between the northern and southern parts of the bay. The density of species and species diversity were high throughout the year in the southern part of the bay, whereas in the northern part of the bay species diversity was low and defaunation occurred in August. At this time, bottom hypoxia due to temperature and salinity stratification, and high concentrations of nutrients, chlorophyll a, and organic matter in the water column and/or sediment, dominated the northern part of the bay. In October, bottom hypoxia was less severe but was still present in the northern part of the bay, and recolonization by mobile fishes and sessile mussels occurred. Multivariate analyses of the megabenthic community and environmental parameters in August showed the spatial pattern of the community could be explained by concentrations of dissolved oxygen and particulate organic carbon in the bottom water, and total sulfide and total organic carbon in the sediments. In particular, impairment of the biota in the northern area could be explained by the threshold concentrations of dissolved oxygen <1.7 mL L⁻¹ and total organic carbon >20.3 mg g⁻¹.     

The distribution characteristics of β-propeller phytase genes in rhizosphere sediment provide insight into species specialty from phytic mineralization in subtropical and tropical seagrass ecosystems

Seagrass meadows have seriously deteriorated in recent years. Seagrass associated phytate-mineralizing rhizobacteria potentially have functions related to seagrass nutrition, health and sustainable growth. The β-propeller phytases (BPPs) are the only phytase family in aquatic environments, but there are few studies on the BPP community structure of seagrass. In this study, clone libraries and quantitative PCR (qPCR) assays were used to compare the diversity and abundances of the BPP communities of Halodule endl, Halophila ovalis and Thalassia hemprichii in Xisha and Sanya, and to investigate the distribution characteristics of BPP genes in the rhizosphere sediment, which provedes insight into species specialty from phytic mineralization in subtropical and tropical seagrass ecosystems. The highest diversity of BPP genes was found for Thalassia hemprichii in Sanya Bay. Thalassia hemprichii in Sanya had higher abundances of BPPs, which were linked to Gammaproteobacteria. The BPP community diversity and OTUs of Thalassia hemprichii in Sanya were much higher than those of Thalassia hemprichii on Yongxing Island and Stone Island. The seagrass BPP communities had higher diversity and evenness from sampling sites with more human activity. The qPCR results showed that the abundance of phytate acid-degradating bacteria was approximately three times larger in Thalassia hemprichii rhizosphere sediment samples than in Halodule endl and Halophila ovalis rhizosphere sediment samples. This study highlighted that the diversity and abundances of bacteria genetically encoding BPP in the rhizosphere of Thalassia hemprichii were clearly higher than those of Halodule endl and Halophila ovalis. Further study of microbial phosphorus cycling will provide new insights into seagrass meadow ecosystems.

     

Sublethal and sex-specific cypermethrin effects in toxicity tests with the midge <Emphasis Type="Italic">Chironomus riparius</Emphasis> Meigen

We quantified sublethal and sex-specific cypermethrin effects in experiments (29 days) with the midge Chironomus riparius at different levels of sediment organic matter content (0, 5, and 20%). We found highly significant effects of cypermethrin concentrations and sediment type on emergence, mean development rate, and adult size. For example, emergence/survival rates were 70–100% below 0.8 μg/l and unaffected by organic matter content. At 3.2 μg/l, however, no larvae survived in sediment without organic matter, but survival successively increased to 26 ± 11% in sediment with 20% organic matter. Mean development rates were always higher for males than for females, and significant differences between sexes occurred consistently in controls and in treatments with our lowest concentration of 0.05 μg/l. Sex-specific differences in mean development rate decreased across the cypermethrin gradient, suggesting that male development was affected more than that of females at similar concentrations. We also found an increase in adult size across the concentration gradient in sediments lacking organic matter and suggest an increased feeding activity due to sublethal toxic stress as a probable causal mechanism. We speculate that the observed sex-specific effects on development rates and adult size can have strong repercussions on emergence timing and fecundity, respectively.     

Toxicity and transformation of insecticide fenamiphos to the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis>

This study was conducted to investigate the toxicity of the organophosphate insecticide fenamiphos to earthworms (Eisenia fetida) under laboratory conditions. Earthworms were exposed to soils differing in their physico-chemical properties spiked with fenamiphos at concentrations ranging from 10 to 200 mg kg⁻¹ for a period of 4 weeks. Residues of fenamiphos and its metabolites were determined in both soils and earthworms after 4 weeks of pesticide exposure. Fenamiphos was degraded faster in the alkaline soil than in the neutral and acidic soils. Median lethal concentration of fenamiphos in the neutral soil was 228 mg kg⁻¹ soil. Residues of fenamiphos caused a significant reduction in the biomass of worms, especially the ones exposed to the pesticide in the acidic soil. In vitro experiments suggested that fenamiphos was biotransformed in the earthworms principally to its oxide metabolite. To our knowledge, this is the first study demonstrating the biotransformation of fenamiphos by E. fetida.     

Sediment‐contact and survival of fingernail clams: Implications for conducting short‐term laboratory tests

Porewater toxicity tests have been used as indicators of whole sediment toxicity. However, many species commonly tested in porewater predominately reside in the water column and otherwise have little to no direct contact with sediment and associated porewater. We assessed the feasibility of porewater toxicity tests with fingernail clams Musculium transversum, a benthic macroinvertebrate that inhabits soft bottom sediments and feeds by filtering surface and porewater. Fingernail clams were exposed to water or sediment in a 96 h laboratory test with a 5×2 factorial experimental design. The five treatments included sediments from four sites in the Mississippi River and one sediment‐free control (well water). In all treatments, clams were exposed to the sediments or water either directly (no enclosure) or indirectly (enclosure, suspended above the sediment surface). There were three replicates for each of the ten treatment combinations. Overall, survival of fingernail clams did not vary among the five treatments (p=0.36). In treatments without enclosures, survival of clams in the sediment‐free control was not significantly different (p=0.34) from the sediment‐containing treatments. Survival of clams in the sediment‐free control averaged 85%—suggesting that direct sediment contact is not necessary for survival in short‐term tests. In contrast, survival of clams in the sediment‐containing treatments differed significantly (p=0.03) between exposures with (mean, 77%) and without (mean, 89%) enclosures. Thus, fingernail clams may provide an alternative species for evaluating benthic macroinvertebrates in short‐term laboratory porewater tests. However, more information on their physiological requirements and the development of sublethal endpoints is recommended before their use in tests of longer duration. ©2000 John Wiley & Sons, Inc. Environ Toxicol 15: 23–27, 2000     

Transfer modelling and toxicity evaluation of the effluent from an installation of cleansing and uranium recovery using a battery of bioassays

On July 7, 2008, a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) led to the spillage of uranium in a stream. The acute toxicity of the effluent was evaluated, and compared to the toxicity of uranium nitrate in bioassays using several organisms: Chlamydomonas reinhardtii, Daphnia magna, Chironomus riparius and Danio rerio. A sediment bioassay was also performed on C. riparius using water and sediment sampled along the river. Results showed that effluent EC₅₀ 72 h was 0.65 mg U/l for algae and LC₅₀ 48 h was 1.67 mg U/l for daphnia, while values obtained for uranium nitrate were higher. The LC₅₀ 96 h of effluent to C. riparius was 22.7 mg U/l, similar to value for uranium nitrate; the sediment collected was not toxic to C. riparius larvae. The LOEC of effluent and uranium nitrate on HT₅₀ of D. rerio were similar (0.03 mg U/l), but larvae were more sensitive to uranium nitrate than to effluent. Our results suggest that other substances contained in the effluent could potentially be toxic to wildlife in association with uranium. In parallel, the modelling of the transfers based on uranium measurements in the surface water was used to fill data gaps and assess the impact along the river. These results provided an estimate of exposure conditions that occurred along the river. This approach allowed us to see that the risk to ecosystem during this incident was certainly low and concerned a short period of time, but it could have existed at least for some species.     

Genetic diversity of Centella asiatica in China analyzed by inter-simple sequence repeat (ISSR) markers: combination analysis with chemical diversity

Centella asiatica is an important plant species used in traditional Chinese medicine. To help the efficient use and conservation of this species, the genetic diversity of C. asiatica populations in China was investigated using inter-simple sequence repeat (ISSR) markers. Fourteen natural populations comprising 162 individuals were included to estimate genetic diversity. At the species level, genetic diversity was relatively high (P = 66.33%, H = 0.2183, I = 0.3305). At the population level, the genetic diversity of JH (Jinhua, Zhejiang Province, China) and JJ (Jiujiang, Jiangxi Province, China) populations was relatively high (P = 43.88%, 38.78%, H = 0.1610, 0.1301, I = 0.2376, 0.1957, respectively), whereas the genetic diversity of GA (Guang’an, Sichuan Province, China) and EM (E’mei, Sichuan Province, China) was relatively low (P = 10.2%, 5.1%, H = 0.0383, 0.0211, I = 0.0570, 0.0309, respectively). On the basis of Nei’s G _st value, more genetic differentiation among populations was determined (G _st = 0.6573). In addition, the 14 populations were clustered into four groups in view of abundant ISSR data, which further defined the genetic relationship among populations. Interestingly, the genetic clustering result was similar to previous chemical clustering results based on high-performance liquid chromatography (HPLC) data, which would also classify the 14 populations into four groups. Thus, we combined the clustering results and compared their difference. The combined analysis and genetic diversity data provide a scientific basis for conserving populations of relatively high genetic diversity such as JH and JJ populations and establishing good agricultural practices (GAP) for C. asiatica.     

Synthesis,Antibacterial Activities,and 3D‐QSAR of Sulfone Derivatives Containing 1, 3, 4‐Oxadiazole Moiety

A series of sulfone derivatives containing 1, 3, 4‐oxadiazole moiety were prepared and evaluated for their antibacterial activities by the turbidimeter test. Most compounds inhibited growth of Ralstonia solanacearum (R. solanacearum) from tomato and tobacco bacterial wilt with high potency, among which compounds 5a and 5b exhibited the most potent inhibition against R. solanacearum from tomato and tobacco bacterial wilts with EC₅₀ values of 19.77 and 8.29 μg/mL, respectively. Our results also demonstrated that 5a, 5b , and a number of other compounds were more potent than commercial bactericides Kocide 3000 and Thiodiazole Copper, which inhibited R. solanacearum from tomato bacterial wilt with EC₅₀ values of 93.59 and 99.80 μg/mL and tobacco bacterial wilt with EC₅₀ values of 45.91 and 216.70 μg/mL, respectively. The structure–activity relationship (SAR) of compounds was studied using three‐dimensional quantitative structure–activity relationship (3D‐QSAR) models created by comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) based on compound bioactivities against tomato and tobacco bacterial wilts. The 3D‐QSAR models effectively predicted the correlation between inhibitory activity and steric–electrostatic properties of compounds.