Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

Immediate effects of pyridazinone herbicides on photosynthetic electron transport in algal systems

Pyridazinone herbicides, SANDOZ 9785 (4-chloro-5-dimethylamino2-phenyl-3-(2H) pyridazinone), SANDOZ 9789 (4-chloro-5 (methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) and SANDOZ 6706 (4-chloro-5-(methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) inhibited photosystem II electron transport inChlorella protothecoides, when the herbicides were added to the assay medium. The inhibitory eficiency varied with the algal species and the nature of substitution of pyridazinones. Using 3 algal systemsviz., Chlorella, Scenedesmus andAnacystis, the I₅₀ value of for the inhibition of photosynthesis of 3 substituted pyridazinones (SANDOZ 9785, SANDOZ 6706 and SANDOZ 9789) were determined. SANDOZ 9789 was found to be the weakest inhibitor of photosystem II electron transport (H₂O→ benzoquinone) as compared to SANDOZ 9785 and SANDOZ 6706. In general, the order of inhibition could be given as SANDOZ 6706 >- SANDOZ 9785 > SANDOZ 9789. The I₅₀ value of photosynthetic particles obtained fromChlorella cells was similar to that of whole cells, suggesting that the cell wall ofChlorella did not act as a barrier for the herbicide action. Studies on the light intensity dependence of SANDOZ 9785 inhibition of electron transport (H₂O→ benzoquinone) showed that the light-dependent portion of the curve was more sensitive than the light independent portion of the curve. It is suggested that the site of action was on the reducing side of photosystem II.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Functional analysis of the two homologous psbA gene copies in Synechocystis PCC 6714 and PCC 6803

The cyanobacteria Synechocystis 6803 and 6714 contain three genes (psbA) coding for the D₁ protein. This protein is an essential subunit of photosystem II (PSII) and is the target for herbicides. We have used herbicide-resistant mutants to study the role of the two homologous copies of the psbA genes in both strains (the third copy is not expressed). Several herbicide resistance mutations map within the psbAI gene in Synechocystis 6714 (G. Ajlani et al.), Plant Mol. Biol. 13 (1989): (469–479). We have looked for mutations in copy II. Results show that in Synechocystis 6714, only psbAI contains herbicide resistance mutations. Relative expression of psbAI and psbAII has been measured by analysing the proportions of resistant and sensitive D₁ in the thylakoid membranes of the mutants. In normal growth conditions, 95% resistant D₁ and 5% sensitive D₁ were found. In high light conditions, expression of psbAII was enhanced, producing 15% sensitive D₁. This enhancement is specifically due to high light and not to the decrease of D₁ concentration caused by photoinhibition. Copy I of Synechocystis 6714 corresponds to copy 2 of Synechocystis 6803 since it was always psbA2 which was recombined in Synechocystis 6803 transformants. PSII of the transformant strains was found to be 95% resistant to herbicides as in resistant mutants of Synechocystis 6714.     

Yessotoxin detected in mussel (Mytilus californicus) and phytoplankton samples from the U.S. west coast

Yessotoxin (YTX) was detected in an algal sample and two mussel samples (0.07–0.10 μg g⁻¹) collected from Scripps Pier in La Jolla, California during a bloom of Lingulodinium polyedrum. Mussel samples collected from Monterey Bay, California also contained measurable YTX (levels up to 0.06 μg g⁻¹) in samples obtained during a 6-month (weekly) sampling period. Gonyaulax spinifera and L. polyedrum were identified in background concentrations in Monterey Bay during the time of contamination. An algal sample from Washington coastal waters collected during non-bloom conditions also contained YTX, possibly originating from Protoceratium reticulatum.Three strains of L. polyedrum (CCMP1931, CCMP1936, 104A) isolated from southern California coastal waters and one strain of G. spinifera (CCMP409) isolated from Maine were tested for YTX production using two methods, competitive ELISA and liquid chromatography–mass spectrometry (LC–MS). The ELISA method detected YTX in the particulate phase in two of three L. polyedrum strains. The LC–MS method did not detect YTX in the particulate or dissolved phase of any of the strains.To our knowledge, this is the first study to test and confirm YTX in environmental samples from California and Washington coastal waters. It is highly likely that L. polyedrum was responsible for the YTX contamination in the southern California samples. Future research needs to conclusively determine the biological origin(s) of YTX contamination in central California and Washington coastal waters.     

Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions

A pot experiment was conducted in the green house to investigate the establishment of phosphate solubilizing strains of Azotobacter chroococcum, including soil isolates and their mutants, in the rhizosphere and their effect on growth parameters and root biomass of three genetically divergent wheat cultivars (Triticum aestivum L.). Five fertilizer treatments were performed: Control, 90 kg N ha^—1, 90 kg N + 60 kg P₂O₅ ha^—1, 120 kg N ha^—1 and 120 kg N + 60 kg P₂O₅ ha^—1. Phosphate solubilizing and phytohormone producing parent soil isolates and mutant strains of A. chroococcum were isolated and selected by an enrichment method. In vitro phosphate solubilization and growth hormone production by mutant strains was increased compared with soil isolates. Seed inoculation of wheat varieties with P solubilizing and phytohormone producing A. chroococcum showed better response compared with controls. Mutant strains of A. chroococcum showed higher increase in grain (12.6%) and straw (11.4%) yield over control and their survival (12—14%) in the rhizosphere as compared to their parent soil isolate (P4). Mutant strain M37 performed better in all three varieties in terms of increase in grain yield (14.0%) and root biomass (11.4%) over control.     

Microalgae fiber optic biosensors for herbicide monitoring using sol–gel technology

Three microalgal species (Dictyosphaerium chlorelloides (D.c.), Scenedesmus intermedius (S.i.) and Scenedesmus sp. (S.s.)) were encapsulated in silicate sol–gel matrices and the increase in the amount of chlorophyll fluorescence signal was used to quantify simazine. Influence of several parameters on the preparation of the sensing layers has been evaluated: effect of pH on sol–gel gelation time; effect of algae density on sensor response; influence of glycerol (%) on the membrane stability. Long term stability was also tested and the fluorescence signal from biosensors remained stable for at least 3 weeks. D.c. biosensor presented the lowest detection limits for simazine (3.6 μg L⁻¹) and the broadest dynamic calibration range (19–860 μg L⁻¹) with IC₅₀ 125 ± 14 μg L⁻¹. Biosensor was validated by HPLC with UV/DAD detection. The biosensor showed response to those herbicides that inhibit the photosynthesis at photosystem II (triazines: simazine, atrazine, propazine, terbuthylazine; urea based herbicides: linuron). However, no significant increases of fluorescence response was obtained for similar concentrations of 2,4-D (hormonal herbicide) or Cu(II). The combined use of two biosensors that use two different genotypes, sensitive and resistant to simazine, jointly allowed improving microalgae biosensor specificity.     

On the control of HAB species using low biosurfactant concentrations

Biosurfactants have been suggested as a method to control harmful algal blooms (HABs), but warrant further and more in-depth investigation. Here we have investigated the algicidal effect of a biosurfactant produced by the bacterium Pseudomonas aeruginosa on five diverse marine and freshwater HAB species that have not been tested previously. These include Alexandrium minutum (Dinophycaee), Karenia brevis (Dinophyceae), Pseudonitzschia sp. (Bacillariophyceae), in marine ecosystems, and Gonyostomum semen (Raphidophyceae) and Microcystis aeruginosa (Cyanophyecae) in freshwater. We examined not only lethal but also sub-lethal effects of the biosurfactant. In addition, the effect of the biosurfactant on Daphnia was tested. Our conclusions were that very low biosurfactant concentrations (5 μg mL⁻¹) decreased both the photosynthesis efficiency and the cell viability and that higher concentrations (50 μg mL⁻¹) had lethal effects in four of the five HAB species tested. The low concentrations employed in this study and the diversity of HAB genera tested suggest that biosurfactants may be used to either control initial algal blooms without causing negative side effect to the ecosystem, or to provoke lethal effects when necessary.     

Effect of Aeolosoma sp. (Aphanoneura: Aeolosomatidae) on the Population Dynamics of Selected Cladoceran Species

Although oligochaete worms naturally coexist with cladocerans in many shallow freshwater ponds and lakes, their influence on the latter is not well established. In this work we studied the effect of Aeolosoma sp. on the population growth of Alona rectangula, Ceriodaphnia dubia, Daphnia pulex, Macrothrix triserialis and Moina macrocopa. Population growth studies were conducted at one algal food density (1 × 10⁶cells ml^–1 of Chlorella vulgaris). The experimental design was similar for all five cladoceran species, where we used 100 ml capacity transparent jars containing 50 ml of EPA medium with the desired algal density and three replicates for each treatment. The test medium was changed daily and fresh algal food was added. The initial density of each of the cladoceran species in the population growth studies was 0.4 ind ml^–1 while that of the worms 1.0 ind ml^–1. Following inoculation, we estimated daily the number of cladocerans and the worms for duration of 21 days. Regardless of the presence of worms, Moina macrocopa and Macrothrix triserialis showed rapid population growth while A. rectangula took more than 2 weeks to reach peak abundances. With the exception of M. triserialis, all the other our cladoceran species declined in the presence of Aeolosoma sp. The lowest peak population density (about 1 ind ml^–1) was observed for M. triserialisin controls. The remaining species had peak densities of about 3–5 ind ml^–1. The rates of population increase per day varied from 0.03 to 0.19 depending on the cladoceran taxa and the treatment. In general we found that pelagic taxa were more adversely affected by the presence of the worms than were the littoral cladocerans.     

Salimicrobium salexigens sp. nov., a moderately halophilic bacterium from salted hides

Two Gram-positive, moderately halophilic bacteria, designated strains 29CMI^T and 53CMI, were isolated from salted hides. Both strains were non-motile, strictly aerobic cocci, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–12.5% [w/v] NaCl), between pH 5.0 and 10.0 (optimal growth at pH 7.5) and at temperatures between 15 and 40 °C (optimal growth at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed a similarity of 98.7% and were closely related to species of the genus Salimicrobium, within the phylum Firmicutes. Strains 29CMI^T and 53CMI exhibited 16S rRNA gene sequence similarity values of 97.9–97.6% with Salimicrobium album DSM 20748^T, Salimicrobium halophilum DSM 4771^T, Salimicrobium flavidum ISL-25^T and Salimicrobium luteum BY-5^T. The DNA G+C content was 50.7 mol% and 51.5 mol% for strains 29CMI^T and 53CMI, respectively. The DNA–DNA hybridization between both strains was 98%, whereas the values between strain 29CMI^T and the species S. album CCM 3517^T, S. luteum BY-5^T, S. flavidum ISL-25^T and S. halophilum CCM 4074^T were 45%, 28%, 15% and 10%, respectively, showing unequivocally that strains 29CMI^T and 53CMI constitute a new genospecies. The major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0, iso-C_15:0 and iso-C_14:0. The main respiratory isoprenoid quinone was MK-7, although small amounts of MK-6 were also found. The polar lipids of the type strain consist of diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid and one glycolipid. The peptidoglycan type is A1γ, with meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of the phylogenetic analysis, and phenotypic, genotypic and chemotaxonomic characteristics, we propose strains 29CMI^T and 53CMI as a novel species of the genus Salimicrobium, with the name Salimicrobium salexigens sp. nov. The type strain is 29CMI^T (=CECT 7568^T = JCM 16414^T = LMG 25386^T).     

Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species

A series of experiments was conducted to examine effects of four strains of the estuarine dinoflagellate, Pfiesteria shumwayae, on the behavior and survival of larval and adult shellfish (bay scallop, Argopecten irradians; eastern oyster, Crassostrea virginica; northern quahogs, Mercenaria mercenaria; green mussels, Perna viridis [adults only]). In separate trials with larvae of A. irradians, C. virginica, and M. mercenaria, an aggressive predatory response of three strains of algal- and fish-fed P. shumwayae was observed (exception, algal-fed strain 1024C). Larval mortality resulted primarily from damage inflicted by physical attack of the flagellated cells, and secondarily from Pfiesteria toxin, as demonstrated in larval C. virginica exposed to P. shumwayae with versus without direct physical contact. Survival of adult shellfish and grazing activity depended upon the species and the cell density, strain, and nutritional history of P. shumwayae. No mortality of the four shellfish species was noted after 24 h of exposure to algal- or fish-fed P. shumwayae (strains 1024C, 1048C, and CCMP2089) in separate trials at ≤5 × 10³ cells ml⁻¹, whereas higher densities of fish-fed, but not algal-fed, populations (>7–8 × 10³ cells ml⁻¹) induced low (≤15%) but significant mortality. Adults of all four shellfish species sustained >90% mortality when exposed to fish-fed strain 270A1 (8 × 10³ cells ml⁻¹). In contrast, adult M. mercenaria and P. viridis exposed to a similar density of fish-fed strain 2172C sustained <15% mortality, and there was no mortality of A. irradians and C. virginica exposed to that strain. In mouse bioassays with tissue homogenates (adductor muscle, mantle, and whole animals) of A. irradians and M. mercenaria that had been exposed to P. shumwayae (three strains, separate trials), mice experienced several minutes of disorientation followed by recovery. Mice injected with tissue extracts from control animals fed cryptomonads showed no response. Grazing rates of adult shellfish on P. shumwayae (mean cell length ±1 standard error [S.E.], 9 ± 1 μm) generally were significantly lower when fed fish-fed (toxic) populations than when fed populations that previously had been maintained on algal prey, and grazing rates were highest with the nontoxic cryptomonad, Storeatula major (cell length 7 ± 1 μm). Abundant cysts of P. shumwayae were found in fecal strands of all shellfish species tested, and ≤45% of the feces produced viable flagellated cells when placed into favorable culture conditions. These findings were supported by a field study wherein fecal strands collected from field-collected adult shellfish (C. virginica, M. mercenaria, and ribbed mussels, Geukensia demissa) were confirmed to contain cysts of P. shumwayae, and these cysts produced fish-killing flagellated populations in standardized fish bioassays. Thus, predatory feeding by flagellated cells of P. shumwayae can adversely affect survival of larval bivalve molluscs, and grazing can be depressed when adult shellfish are fed P. shumwayae. The data suggest that P. shumwayae could affect recruitment of larval shellfish in estuaries and aquaculture facilities; shellfish can be adversely affected via reduced filtration rates; and adult shellfish may be vectors of toxic P. shumwayae when shellfish are transported from one geographic location to another.     

Using quantitative real-time PCR to study competition and community dynamics among Delaware Inland Bays harmful algae in field and laboratory studies

The Delaware Inland Bays (DIB) have experienced harmful algal blooms of dinoflagellates and raphidophytes in recent years. We used quantitative polymerase chain reaction (QPCR) techniques to investigate the community dynamics of three DIB dinoflagellates (Karlodinium veneficum, Gyrodinium instriatum, and Prorocentrum minimum) and one raphidophyte (Heterosigma akashiwo) at a single site in the DIB (IR-32) in summer 2006 relative to salinity, temperature and nutrient concentrations. We also carried out complementary laboratory culture studies. New primers and probes were developed and validated for the 18S rRNA genes in the three dinoflagellates. K. veneficum, H. akashiwo, and G. instriatum were present in almost all samples throughout the summer of 2006. In contrast, P. minimum was undetectable in late June through September, when temperatures ranged from 20 to 30 °C (average 25.7 °C). Dissolved nutrients ranged from 0.1 to 2.8 μM PO₄³⁻ (median = 0.3 μM), 0.7–30.2 μM NO_x (median = 12.9 μM), and 0–19.4 μM NH₄⁺ (median = 0.7 μM). Dissolved N:P ratios covered a wide range from 2.6 to 177, with a median of 40. There was considerable variability in occurrence of the four species versus nutrients, but in general P. minimum and H. akashiwo were most abundant at higher (>40) N:P ratios and dissolved nitrogen concentrations, while K. veneficum and G. instriatum were most abundant at low dissolved N:P ratios (<20) and dissolved nitrogen concentrations < 10 μM. The semi-continuous laboratory competition experiment used mixed cultures of K. veneficum, P. minimum, and H. akashiwo grown at dissolved N:P ratios of 5, 16, and 25. At an N:P of 16 and 25 P. minimum was the dominant alga at the end of the experiment, even at a temperature that was much higher than that at which this alga was found to bloom in the field (27 °C). P. minimum and H. akashiwo had highest densities in the N:P of 25. K. veneficum grew equally well at all three N:P ratios, and was co-dominant at times at an N:P of 5. H. akashiwo had the lowest densities of the three algae in the laboratory experiment. Laboratory and field results showed both interesting similarities and significant differences in the influences of important environmental factors on competition between these harmful algal species, suggesting the need for more work to fully understand HAB dynamics in the DIB.     

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).     

Relationships among the herbicide and functional sites of acetohydroxy acid synthase from Chlorella emersonii

The properties of acetohydroxy acid synthase (AHAS, EC 4.1.3.18) from wild-type Chlorella emersonii (var. Emersonii, CCAP-211/11n) and two spontaneous sulfometuron methyl (SMM)-resistant mutants were examined. The AHAS from both mutants was resistant to SMM and cross-resistant to imazapyr (IM) and the triazolopyrimidine sulfonanilide herbicide XRD-498 (TP). The more-SMM-resistant mutant had AHAS with altered catalytic parameters (K _m, specificity), but unchanged sensitivity to the feedback inhibitors valine and leucine. The second mutant enzyme was less sensitive to the feedback inhibitors, but had otherwise unchanged kinetic parameters. Inhibition-competition experiments indicated that the three herbicides (SMM, IM, TP) bind in a mutually exclusive manner, but that valine can bind simultaneously with SMM or TP. The three herbicide classes apparently bind to closely overlapping sites. We suggest that the results with C. emersonii and other organisms can all be explained if there are separate binding sites for herbicides, feedback inhibitors and substrates.Abbreviations AHAS acetohydroxy acid synthase - AL acetolactate - AHB acetohydroxybutyrate - IM imazapyr - TP triazolopyrimidine sulfonanilide herbicide XRD-498 - R enzyme specificity - SMM sulfometuron methyl This research was supported in part by the United States — Israel Binational Science Foundation (BSF), Jerusalem, Israel (Grant 86-00205) and the Fund for Basic Research, Israel Academy of Sciences.     

Screening of algal strains for metal removal capabilities

Eight algal species were tested for their ability to remove five toxic metalsduring 30-min exposures to single-metal (1 mg L^-1) solutions at pH7. Efficacy of metal bioremoval varied according to algal species and metal. Al⁺³ was best removed by the thermophilic blue-green alga(cyanobacterium) Mastigocladus laminosus, Hg⁺² and Zn⁺² by the thermophilic and acidophilic red alga Cyanidiumcaldarium, and Cd⁺² by C. caldarium and the green alga Scenedesmus quadricauda. All of these alga/metal combinations resultedin >90% metal removal. However, none of the eight algal speciesremoved more than 10% of Cr⁺⁶. Results indicate that some toxicmetals are more readily removed than others are by algae and that selectionof appropriate strains could potentially enhance bioremoval of specificmetals from wastewater at neutral pH.     

Biodeterioration of some herbal raw materials by storage fungi and aflatoxin and assessment of Cymbopogon flexuosus essential oil and its components as antifungal

Deterioration of raw materials of six medicinal plants viz. Terminalia arjuna, Acorus calamus, Rauvolfia serpentina, Holarrhena antidysenterica, Withania somnifera and Boerhaavia diffusa was examined. Some of the contaminated raw materials were found to be deteriorated by toxigenic strains of Aspergillus flavus and contain aflatoxin B₁ (41.0–95.4 μg kg⁻¹) which is above the permissible limit. Essential oil of Cymbopogon flexuosus and its components was found efficient in checking fungal growth and aflatoxin production. C. flexuosus essential oil absolutely inhibited the growth of A. flavus and aflatoxin B₁ production at 1.3 μl ml⁻¹ and 1.0 μl ml⁻¹ respectively. The individual oil components were more efficacious than the Cymbopogon oil as such which emphasizes masking of their efficacy when combined together. Eugenol exhibited potent antifungal and aflatoxin inhibitory activity at 0.3 μl ml⁻¹ and 0.1 μl ml⁻¹ respectively. Eugenol was found superior over some prevalent synthetic antimicrobials and exhibited broad fungitoxic spectrum against some biodeteriorating moulds. Prospects of exploitation of the oil and its components as acceptable plant based antimicrobials in qualitative as well as quantitative control of biodeterioration of herbal raw materials have been discussed.     

A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

First occurrence of Cochlodinium blooms in Sabah, Malaysia

Harmful algal blooms (HABs) resulting in red discoloration of coastal waters in Sepanggar Bay, off Kota Kinabalu, Sabah, East Malaysia, were first observed in January 2005. The species responsible for the bloom, which was identified as Cochlodinium polykrikoides, coincided with fish mortalities in cage-cultures. Determinations of cell density between January 2005 and June 2006 showed two peaks that occurred in March–June 2005 and June 2006. Cell abundance reached a maximum value of 6 × 10⁶ cells L⁻¹ at the fish cage sampling station where the water quality was characterized by high NO₃–N and PO₄–P concentrations. These blooms persisted into August 2005, were not detected during the north–east monsoon season and occurred again in May 2006. Favorable temperature, salinity and nutrient concentrations, which were similar to those associated with other C. polykrikoides blooms in the Asia Pacific region, likely promoted the growth of this species. Identification of C. polykrikoides as the causative organism was based on light and scanning microscopy, and confirmed by partial 18S ribosomal DNA sequences of two strains isolated during the bloom event (GenBank accession numbers DQ915169 and DQ915170).     

Isolation and characterization of three <Emphasis Type="Italic">Sphingobium</Emphasis> sp. strains capable of degrading isoproturon and cloning of the catechol 1,2-dioxygenase gene from these strains

Three strains of bacteria (designated as YBL1, YBL2, YBL3 respectively) capable of degrading isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, were isolated from the soils of two herbicide plants. Based on the comparative analysis of the 16S rRNA gene, and phenotypic and biochemical characterization, these strains were identified as Sphingobium sp. The optimum conditions for isoproturon degradation by these strains were pH 7.0, and temperature 30°C. Mg²⁺ (1 mM) enhanced the isoproturon degradation rate, while Ni²⁺ and Cu²⁺ (1 mmol l⁻¹) inhibited isoproturon degradation significantly. These three strains also showed the ability to remove the residues of other phenylurea herbicides such as chlorotoluron, diuron and fluometuron in mineral salt culture medium. The N-demethylation was the first step of degradation of dimethylurea-substituted herbicides. Strain YBL1 was found capable of degrading both dimethylurea-substituted herbicides and methoxymethylphenyl-urea herbicides i.e. linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea). Using the PCR method, partial sequences of the catechol 1,2-dioxygenase gene were obtained from these strains.     

Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences

The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed <10% of the cellular N of flagellated cells feeding on algae, but were equal to or greater than phagotrophic N acquisition in cells recently removed from fish cultures. Specific N uptake rates (V, h⁻¹) were higher for cells that were maintained on algal prey for long periods (months) than those that were grown with live fish. However, rates of N uptake on a cellular basis for cells grown on or recently removed from fish were comparable to those maintained on algal prey, likely reflecting differences in the sizes of cells of different physiological condition. Preferences for form of N generally followed a decreasing trend of amino acids > urea > NH₄⁺ > NO₃⁻. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature.