Isolation and characterization of arsenite-oxidizing bacteria from arsenic-contaminated soils in Thailand

Two hundred and eighty-eight arsenic-resistant bacteria were isolated by an enrichment culture method from a total of 69 arsenic-contaminated soil-samples collected from Dantchaeng district in Suphanburi province (47 samples), and from Ron Phiboon district in Nakhon Sri Thammarat province (22 samples), in Central and Southern Thailand, respectively. Twenty-four of the 288 isolated arsenic-resistant bacteria were found to be arsenite-oxidizing bacteria. On the basis of their morphological, cultural, physiological, biochemical and chemotaxonomic characteristics, and supported by phylogenetic analysis based upon their 16S rRNA gene sequences, they were divided into five groups, within the genera Acinetobacter, Flavobacterium, Pseudomonas, Sinorhizobium and Sphingomonas, respectively. Within genera, phylogenetic analysis using the 16S rRNA gene sequences suggested that they were comprised of at least ten species, five isolates being closely related to known bacteria (Acinetobacter calcoaceticus NCCB 22016^T, Pseudomonas plecoglossicida FPC951^T, Ps. knackmussii B13^T, Sinorhizobium morelense Lc04^T, and Sphingomonas subterranea IFO16086^T). The other five proposed species are likely to be new species closely related to Flavobacterium johnsoniae, Sinorhizobium morelense, Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, but this awaits further characterization for confirmation of the taxonomic status. No overlap in isolated species or strains was observed between the two sites. The strain distribution and characterization are described.     

结直肠癌相关致病菌的研究进展

肠道微生物群落与结直肠癌（Colorectal Cancer,CRC）有着十分密切的关系。肠道微生物的群落变化可能会伴随着CRC的发生,而一些有害菌的出现可能是导致CRC的直接原因。其中,具核梭杆菌（Fusobacterium nucleatum）、产肠毒素脆弱拟杆菌（Enterotoxigenic Bacteroides fragilis,ETBF）和pks阳性大肠杆菌（pks⁺ Escherichia coli）与CRC的发生最密切。本综述着重介绍了pks⁺ E. coli及Colibactin的致病原因、对肠道微生物组成的影响、Colibactin的合成及怎样抑制或促进pks⁺ E. coli。同时也对ETBF和F. nucleatum可能的致癌原因、对肠道微生物组成的影响及对二者的促进或抑制做出了介绍。     

Effect of two microalgae concentrations on body size and egg size of the rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis>

The rotifer, Brachionus calyciflorus, was grown with two algae species (Chlorella sp. and Scenedesmus obliquus) at different concentrations (0.1, 1 and 10 × 10⁶ cells ml⁻¹). The body size (lorica biovolume) of individual rotifer and their egg size were measured when the populations were roughly in the exponential phase of population growth. The body size of the rotifers differed significantly (P < 0.05) among the two algae species used, however this effect was not observed for egg size. The body size of rotifers fed on higher densities of Chlorella sp. (10 × 10⁶ cells ml⁻¹) was significantly larger than for those fed on lower and medium densities (0.1 and 1 × 10⁶ cells ml⁻¹). Body size and egg size of rotifers fed with different amounts of Scenedesmus did not differ significantly. The egg size was significantly larger at higher food level of Chlorella. A significantly positive correlation was observed between the adult rotifer body size and their egg size.     

Field metabolic rates and water influxes of two sympatric Gerbillidae:Gerbillus allenbyi andG. pyramidum

Summary Two primarily granivorous rodents of Old World deserts,Gerbillus allenbyi (mean adult body mass=26 g) andG. pyramidum (mean adult body mass=40 g), coexist in sandy habitats in the northwestern Negev desert. Both species are burrow dwellers and are nocturnal; however, in their overall distributions,G. pyramidum occurs in more extreme deserts than doesG. allenbyi. In comparing field metabolic rate (FMR) and water influx of the twoGerbillus species, we considered two alternative hypotheses: (1) given the difference in their overall distributions,G. pyramidum has a lower FMR and water influx thanG. allenbyi, and (2) given the similarity in their diets, and that we worked with sympatric populations, FMR and water influx are similar. The latter alternative proved to be correct. Field metabolic rates in summer were 7.29 kJ · g^-0.51 · day^-1 forG. allenbyi and 7.74 kJ · g^-0.51 · day^-1 forG. pyramidum, values that were 69.3% and 74.5%, respectively, of those predicted for rodents of their body masses. Summer water influx ofG. allenbyi was 0.167 ml · g^-0.90 · day^-1 and that ofG. pyramidum was 0.144 ml · g^-0.90 · day^-1; these values were 79.4% and 68.6%, respectively, of water influxes predicted for rodents of their body masses. When compared allometrically, there were no interspecific differences in any of the measurements.     

Phylogenetic diversity based on <Emphasis Type="Italic">rrs,atpD, recA</Emphasis> genes and 16S–23S intergenic sequence analyses of rhizobial strains isolated from <Emphasis Type="Italic">Vicia faba</Emphasis> and <Emphasis Type="Italic">Pisum sativum</Emphasis> in Peru

In this study 17 isolates from effective nodules of Vicia faba and Pisum sativum var. macrocarpum growing in different soils from Peru were isolated and characterized. The isolates, presenting 11 different RAPD profiles, were distributed in three groups on the basis of their 16S-RFLP patterns. The 16S rRNA gene sequences of strains from 16S-RFLP groups I, II and III were closely related (identities higher than 99.5%) to Rhizobium leguminosarum bv. trifolii DSM 30141 (=ATCC 14480), R. leguminosarum bv. viciae DSM 30132^T and Rhizobium etli CFN42^T (=USDA 9032^T), respectively. The analysis of the 16S–23S intergenic spacer (ITS) and two housekeeping genes, atpD and recA, confirmed the identification of strains from group I, however those from groups II and III were phylogenetically divergent to strains DSM 30132^T and CFN42^T. These results support the fact that the 16S rRNA gene is not adequate for identification at species level within genus Rhizobium and suggest the existence of putative new species within the phylogenetic group of R. leguminosarum. They also confirm the need of a taxonomic revision of R. leguminosarum since the reference strains of the three biovars included in this study are phylogenetically divergent according to their ITS, atpD and recA gene sequences.     

Natronolimnobius baerhuensis gen. nov., sp. nov. and Natronolimnobius innermongolicus sp. nov., novel haloalkaliphilic archaea isolated from soda lakes in Inner Mongolia, China

Three novel isolates of haloalkaliphilic archaea, strains IHC-005^T, IHC-010, and N-1311^T, from soda lakes in Inner Mongolia, China, were characterized to elucidate their taxonomic positions. The three strains were aerobic, Gram-negative chemoorganotrophs growing optimally at 37–45°C, pH 9.0–9.5, and 15–20% NaCl. Cells of strains IHC-005^T/IHC-010 were motile rods, while those of strain N-1311^T were non-motile pleomorphic flats or cocci. The three strains contained diphytanyl and phytanyl-sesterterpanyl diether derivatives of phosphatidylglycerol and phosphatidylglycerophosphate methyl ester. No glycolipids were detected. On phylogenetic analysis of 16S rRNA gene sequences, they formed an independent cluster in the Natro group of the family Halobacteriaceae. Comparison of their morphological, physiological, and biochemical properties, DNA G+C content and 16S rRNA gene sequences, and DNA-DNA hybridization study support the view that strains IHC-005^T/IHC-010 and strain N-1311^T represent separate species. Therefore, we propose Natronolimnobius baerhuensis gen. nov., sp. nov. for strains IHC-005^T (=CGMCC 1.3597^T =JCM 12253^T)/IHC-010 (=CGMCC 1.3598=JCM 12254) and Natronolimnobius innermongolicus sp. nov. for N-1311^T (=CGMCC 1.2124^T =JCM 12255^T).     

Chromosome-encoded inducible copper resistance inPseudomonas strains

NinePseudomonas strains were selected by their high copper tolerance from a population of bacteria isolated from heavy-metal polluted zones. Copper resistance (Cu ^r ) was inducible by previous exposure of cultures to subinhibitory amounts of copper sulfate. All nine strains possessed large plasmids, but transformation and curing results suggest that Cu ^r is conferred by chromosomal genes. Plasmid-lessPseudomonas aeruginosa PAO-derived strains showed the same level of Cu ^r as environmental isolates and their resistance to copper was also inducible. Total DNA from the environmentalPseudomonas, as well as fromP. aeruginosa PAO strains, showed homology to a Cu ^r P. syringae cop probe at low-stringency conditions but failed to hybridize at high-stringency conditions.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Stage-specific mortality of Rhagoletis indifferens (Diptera: Tephritidae) exposed to three species of Steinernema nematodes

Mortality of larval, pupal, and adult western cherry fruit fly, Rhagoletis indifferens (Tephritidae) exposed to the steinernematid nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema intermedium, was determined in the laboratory and field. Larvae were the most susceptible stage, with mortality in the three nematode treatments ranging from 62 to 100%. S. carpocapsae and S. feltiae were equally effective against larvae at both 50 and 100 infective juveniles (IJs)/cm². S. intermedium was slightly less effective against larvae than the other two species. Mortalities of R. indifferens larvae at 0, 2, 4, and 6 days following their introduction into soil previously treated with S. carpocapsae and S. feltiae at 50 IJs/cm² were 78.6, 92.5, 95.0, and 77.5% and 87.5, 52.5, 92.5, and 70.0%, respectively, and at 100 IJs/cm² were 90.0, 92.0, 100.0, and 84.0% and 90.0, 50.0, 42.0, and 40.0%, respectively. There was no decline in mortality caused by S. carpocapsae as time progressed, whereas there was in one test with S. feltiae. Larval mortalities caused by the two species were the same in a 1:1:1 vermiculite:peat moss:sand soil mix and a more compact silt loam soil. In the field, S. carpocapsae and S. feltiae were equally effective against larvae. Pupae were not infected, but adult flies were infected by all three nematode species in the laboratory. S. carpocapsae was the most effective species at a concentration of 100 IJs/cm² and infected 11–53% of adults that emerged. The high pathogenicity of S. carpocapsae and S. feltiae against R. indifferens larvae and their persistence in soil as well as efficacy in different soil types indicate both nematodes hold promise as effective biological control agents of flies in isolated and abandoned lots or in yards of homeowners.     

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

Summary Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA ⁺ uvr ⁺bacteria, plasmid pIC80, mucAB ⁺mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC ⁺. A similar result was obtained in lexA51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA ⁺ uvrB5 bacteria, plasmid pSE117,umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These nagative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA ⁺ uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.     

Airborne Ascomycotina on the island of Crete: Seasonal patternsbased on an 8-year volumetric survey

An 8-year study was conducted on the island of Crete in order to identify airborne ascospores and to determine their seasonal pattern. A Burkard 7-day, volumetric spore-trap was continuously operated in the city of Irakleion – located in the center of the island – from 1994 through 2001. Relatively „high” ascospore counts (20 – 48 spores/m ³) were obtained from mid-spring through summer, while the rest of the year exhibited lower activity (8–16 spores/m³). The predominant ascospores identified were those of Leptosphaeria and Chaetomium; their concentrations varied from 1 or 2 spores up to a few dozens of spores/m³. Other spores encountered sporadically were: Ascobolus, Endophragmiella, Didymella, Diatrypaceae, Leptosphaerulina, Massaria, Pleospora, Sporormiella, Xylaria. The mean daily concentration of all identified ascospores was 30/m³ for the entire study period, corresponding to 13.9% of the total fungal load. Ascospores have been recognized as important inhalant allergens and have been implicated as contributing to symptoms of both rhinitis and asthma.     

大型海藻龙须菜抽提液对褶皱臂尾轮虫生命表参数的影响

大型海藻富含多种活性物质,具有抗衰老等生物活性;轮虫是良好的潜在抗衰老研究模式生物。本研究以褶皱臂尾轮虫(Brachionus plicatilis)作为实验对象,研究了不同浓度的大型海藻龙须菜抽提液(0,250,500,750,1000 mg/L)和不同浓度的食物(蛋白核小球藻和普通小球藻)对褶皱臂尾轮虫生命表参数的影响。结果表明:与对照组相比,食物浓度为1.0×10~6个/mL蛋白核小球藻时,不同浓度龙须菜抽提液对轮虫产卵数、平均寿命、净生长率以及世代时间有显著促进效应(P0.05);轮虫平均产卵数及寿命在龙须菜抽提液浓度750 mg/L处达到最高,分别为16只和13.9d(P0.05)。食物浓度为2.0×10~6个/mL普通小球藻时,轮虫平均产卵数和寿命在抽提液浓度为500 mg/L处达到最高,分别为16只和13.6d(P0.05),轮虫平均寿命和净生长率均有显著提高(P0.05)。相同龙须菜抽提液浓度下,食物浓度为1.0×10~6个/mL蛋白核小球藻下轮虫的净生长率、世代时间均显著高于食物浓度为2.0×10~6个/mL蛋白核小球藻培养的轮虫(P0.05);食物浓度为2.0×10~6个/mL时,普通小球藻培养轮虫的净生长率和世代时间均显著高于蛋白核小球藻实验组(P0.05)。交互作用分析显示,龙须菜抽提液与小球藻的交互作用对褶皱臂尾轮虫的内禀增长率有显著影响(P0.05)。研究结果表明,大型海藻龙须菜抽提液对褶皱臂尾轮虫的生长与生殖有促进作用,延长轮虫寿命。     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.     

The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils

Four bacterial isolates were examined for their ability to increase the availability of water soluble Cu, Cr, Pb and Zn in soils and for their effect on metals uptake by Zea mays and Sorghum bicolor. Random Amplified Polymorphic DNA (RAPD) analysis was used to show that the bacterial cultures were genetically diverse. Bacterial isolates S3, S28, S22 and S29 had 16S rRNA gene sequences that were most similar to Bacillus subtilis, Bacillus pumilus, Pseudomonas pseudoalcaligenes and Brevibacterium halotolerans based on 100% similarity in their 16S rDNA gene sequence, respectively. Filtrate liquid media that had supported B. pumilus and B. subtilis growth significantly increased Cr and Cu extraction from soil polluted with tannery effluent and from Cu-rich soil, respectively, compared to axenic media. The highest concentrations of Pb (0.2 g kg⁻¹), Zn (4 g kg⁻¹) and Cu (2 g kg⁻¹) were accumulated in shoots of Z. mays grown on Cu-rich soil inoculated with Br. halotolerans. The highest concentration of Cr (5 g kg⁻¹) was accumulated in S. bicolor roots grown in tannery-effluent-polluted soil inoculated with a mixed inoculum of bacterial strains. These results show that bacteria play an important role in increasing metal availability in soil, thus enhancing Cr, Pb, Zn and Cu accumulation by Z. mays and S. bicolor.     

Nocardia rhamnosiphila sp. nov., isolated from soil

In this study two actinomycete strains were isolated in Cape Town (South Africa), one from a compost heap (strain 202GMO^T) and the other from within the fynbos-rich area surrounded by the horseracing track at Kenilworth Racecourse (strain C2). Based on 16S rRNA gene sequence BLAST analysis, the strains were identified as members of the genus Nocardia. Phylogenetic analysis showed that the strains clustered together and are most closely related to Nocardia flavorosea NRRL B-16176^T, Nocardia testacea JCM 12235^T, Nocardia sienata IFM 10088^T and Nocardia carnea DSM 43397^T. This association was also supported by gyrB based phylogenetic analysis. The results of DNA–DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of both strains 202GMO^T and C2 from related species. However, their high DNA relatedness showed that they belong to the same species. Strain 202GMO^T was selected as the type strain to represent this novel species, for which the name Nocardia rhamnosiphila is proposed (=DSM 45147^T = NRRL B-24637^T).     

Five novel elements involved in the regulation of mitosis in fission yeast

Summary Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 weel ^ts cdc25^ts triple mutant strains. These genes were designated wisl ⁺ –wis5⁺for win suppressing, and do not correspond to winl ⁺ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25 ^ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wisl ⁺ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4 ⁺ appears to be a specific suppressor of the winl-1 mutation. wis2 ⁺ and wis3 ⁺ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1 ^ts and cdc25 ^ts, suggesting that the wis2 ⁺ and wis3 ⁺ products may interact with elements central to the mitotic control.     

Habitat conditions of the phytocoenoses dominated by Luronium natans (L.) Rafin in Poland

Phytosociological and habitat studies of the phytocoenoses of Luronium natans have been conducted. The present results were compared with data on L. dortmanna and I. lacustris. It is demonstrated that the community of L. natans differs from the other two communities with respect to habitat conditions despite the fact that they have been reported to occur jointly and alongside in Lobelia lakes. It appears that significant differences between the communities are found not only regarding their waters, but also their substrates. L. natans dominated phytocoenoses are confined to oligotrophic, extremely soft waters, markedly poor in Ca²⁺, but richer in Na⁺ and SO₄ ^2- than those of Lobelia and Isoëtes. Luronium natans develops best on acidic, highly hydrated substrates rich in organic matter, NO₃ ^– and total N. The results obtained indicate that L. natans and the phytocoenoses formed by it are characterized by their narrow ecological amplitude in Poland as opposed to those occurring in western Europe, which tolerate a relatively wide range of habitats. The present findings confirm the data from numerous works, which point to the weak competitive ability of the species compared with species typical of eutrophic waters.     

Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh

Summary 600,000 seedlings ofAlnus crispa were inoculated with a 111 mixture of theFrankia strains ACN1 ^AG , AGN1 _exo ^AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod^–) with Frankiae. This selected individual AC-4 (Nod^–) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) withFrankia werein vitro propagated. The different clones ofA. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7Frankia strains indicated that the clone AC-4 (Nod^–) was non-nodulating with 6 of the 7Frankia strains tested. One strain,Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod⁺) and AC-5 (Nod⁺). Morphological observations of the roots of AC-4 (Nod^–) indicated that this clone had few and abnormally short root hairs.     

Interactions and effects of multiple biological control insects on diffuse and spotted knapweed in the Front Range of Colorado

Abundances and interactions among biological control insects and their effects on target invasive plants were monitored within the flower heads and roots of diffuse knapweed, Centaurea diffusa, and in spotted knapweed, Centaurea stoebe, along the Colorado Front Range. Flower weevils, (Larinus species) and root-feeders (Cyphocleonus achates and Sphenoptera jugoslavica) were released on knapweed that already supported biological control gall flies (Urophora species). At a single monitoring site, seed production by C. diffusa declined from 4400 seeds m⁻² in 1997 to zero seeds m⁻² on the monitoring sites in 2006, while the flowering stem density of C. diffusa declined from a peak of almost 30 stems m⁻² in 2000 to zero stems m⁻² in 2006. The average abundance of Urophora and Larinus in flower heads fluctuated independently during the 2001–2006 interval, while the relative abundance of C. achates and S. jugoslavica in roots exhibited a weak inverse relationship that appeared driven by climate effects. The relative abundance of insects on a population of C. stoebe was monitored for five years as Larinus species and C. achates became established on spotted knapweed that already supported Urophora species. Spotted knapweed seed production on our monitoring site declined from 4600 seeds m⁻² in 2003 to zero seeds m⁻² in 2006. Unlike C. diffusa, substantial numbers of rosettes of C. stoebe remained present. Larinus consumed almost all Urophora encountered in C. diffusa, and consumed about 40% of the Urophora in co-infested flower heads of C. stoebe (ca. 10–15% of the total Urophora population). No negative correlations between the relative densities of flower head and root-feeding insects were observed. The effects of these insects on target plants have produced results consistent with the ‘cumulative stress hypothesis’ for biological control of Centaurea species.