Correlation of ecological factors and allozymic variations with resistance toErysiphe graminis hordei inHordeum spontaneum in Israel: Patterns and application

The association of ecological factors and allozymic markers of wild barley,Hordeum spontaneum, with genotypes varying in resistance to 3 cultures of the pathogenErysiphe graminis hordei, which incites the disease powdery mildew of barley, were explored theoretically and practically. The study involved 275 accessions comprising 16 populations largely representing the ecological range ofH. spontaneum in Israel. From earlier studies of allozymic variation and disease resistance it now becomes apparent that genetic polymorphisms for resistance toE. graminis hordei are structured geographically, and are predictable by climatic as well as allozymic variables. Three-variable combinations of temperature and water factors explain significantly 0.32 of the spatial variance in disease resistance between localities. Also, several allozyme genotypes, singly or in combination, are significantly associated with disease resistance. A high correlation was found between the standard deviation of infection types of the culture of the pathogen from Israel, and allozymic polymorphism,P (r_s = 0.86, p < 0.001). Consequently, the IsraelH. spontaneum populations, growing in the center of diversity of the species, contain large amounts of unexploited disease resistance polymorphism. These could be effectively screened and utilized for producing resistant barley varieties by using ecological factors and allozymic variants as guidelines.     

AB-QTL analysis in spring barley. I. Detection of resistance genes against powdery mildew, leaf rust and scald introgressed from wild barley

The objective of this study was to map new resistance genes against powdery mildew (Blumeria graminis f. sp. hordei L.), leaf rust (Puccinia hordei L.) and scald [Rhynchosporium secalis (Oud.) J. Davis] in the advanced backcross doubled haploid (BC₂DH) population S42 derived from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). Using field data of disease severity recorded in eight environments under natural infestation and genotype data of 98 SSR loci, we detected nine QTL for powdery mildew, six QTL for leaf rust resistance and three QTL for scald resistance. The presence of the exotic QTL alleles reduced disease symptoms by a maximum of 51.5, 37.6 and 16.5% for powdery mildew, leaf rust and scald, respectively. Some of the detected QTL may correspond to previously identified qualitative (i.e. Mla) and to quantitative resistance genes. Others may be newly identified resistance genes. For the majority of resistance QTL (61.0%) the wild barley contributed the favourable allele demonstrating the usefulness of wild barley in the quest for resistant cultivars.     

Effect of soil-applied NPK fertilizers on severity of black spot disease (Alternaria brassicae) and yield of oilseed rape

Effect of soil application of eight combinations of NPK fertilizers on the severity of black spot disease (BSD), caused by Alternaria brassicae (Sacc.) Berk., and yield of short duration oilseed rape (Brassica campestris L) were investigated under both pot and field conditions in 1987–88, 1988–89 and 1990–91. The severity of BSD was significantly greater (36–48%) on plants grown in ground treated with NP (N 90 kg ha^–1+P 40 kg ha^–1) applied as urea and single superphosphate respectively than on plants from the unfertilized control (NoPoKo) (o). However, the severity of BSD was significantly smaller (25–33%) when K (40 kg ha^–1) was applied as muriate of potash than in plants from control and NP treatments. The effect of NK (N 90 kg ha^–1+K 40 kg ha^–1) in decreasing the severity of BSD was increasingly more pronounced than the effects of PK (P 40 kg ha^–1+K 40 kg ha^–1), NP and K (40 kg ha^–1) applications. The decrease in the severity of BSD due to K was due to increased production in plants of phenolics which inhibited conidial germination and decreased sporulation of A. brassicae.The decrease in the severity of BSD due to NK application gave consistently increased seed yield 68% more than those of control and other treatments. The K-fertilized plants also showed increased resistance to lodging, increased 1000-seed weight and decreased seed infection. Seeds obtained from K-fertilized plants showed good seed germinability and vigorous seeding growth.     

The effect of small-scale environmental changes on disease incidence and severity in a natural plant-pathogen interaction

Summary The incidence and severity of Rhynchosporium secalis infections were assessed in a large population of Hordeum leporinum. Transects were set out in four directions from five trees to determine the effect of shading. Under the tree canopy 60.3% of H. leporinum plants were infected while only 11.2% were infected away from the canopy. Disease severity, on those plants which were infected, was higher under the canopy (mean 12.4% and 13.0% leaf area diseased for the flag and first leaves, respectively) than away from the canopy (means of 7.8% and 5.0% for the flag and first leaves respectively). Plants under the tree canopy contained on average 23% more nitrogen, raising the possibility that the susceptibility of the host changed in response to nitrogen levels. However, the observed pattern is also consistent with the hypothesis that shade-associated changes in the environment enhanced the ability of the pathogen to infect and develop on the host. The data clearly demonstrate the importance of small-scale environmental factors on natural host-pathogen interactions. These environmental factors may cause differential selection for disease resistance within a host population, which may ultimately lead to the formation of sub-populations with differing levels of resistance.     

Annual productivity of Spirulina (Arthrospira) and nutrient removal in a pig wastewater recycling process under tropical conditions

An evaluation was made of the annual productivity of Spirulina (Arthrospira) and its ability to remove nutrients in outdoor raceways treating anaerobic effluents from pig wastewater under tropical conditions. The study was based at a pilot plant at La Mancha beach, State of Veracruz, Mexico. Batch or semi-continuous cultures were established at different seasons during four consecutive years. The protein content of the harvested biomass and the N and P removal from the ponds were also evaluated. Anaerobic effluents from digested pig waste were added in a proportion of 2% (v/v) to untreated sea-water diluted 1:4 with fresh water supplemented with 2 g L^–1 sodium bicarbonate, at days 0, 3 and 5. A straight filament strain of Spirulina adapted to grow in this complex medium was utilized. A pH value 9.5 ± 0.2 was maintained. The productivity of batch cultures during summer 1998 was significantly more with a pond depth of 0.10 m than with a depth 0.065 m. The average productivity of semi-continuous cultures during summer 1999 was 14.4 g m^–2 d^–1 with a pond depth of 0.15 m and 15.1 g m^–2 d^–1 with a depth of 0.20 m. The average annual productivity for semi-continuous cultures operating with depths of 0.10 m for winter and 0.15 and 0.25 m for the rest of the year, was 11.8 g m^–2 d^–1. This is the highest value reported for a Spirulina cultivation system utilising sea-water. The average protein content of the semi-continuous cultures was 48.9% ash-free dry weight. NH₄-N removal was in the range 84–96% and P removal in the range of 72–87%, depending on the depth of the culture and the season.     

Fusarium head blight biological control with Lysobacter enzymogenes strain C3

Fusarium head blight (FHB), caused by Fusarium graminearum (= Gibberella zeae), is a destructive disease of wheat for which biological controls are needed. Lysobacter enzymogenes strain C3, a bacterial antagonist of fungal pathogens via lytic enzymes and induced resistance, was evaluated in this study for control of FHB. In greenhouse experiments, chitin broth cultures of C3 reduced FHB severity to <10% infected spikelets as compared to >80% severity in the controls in some experiments. C3 broth cultures heated to inactivate cells and lytic enzymes, but retaining the elicitor factor for induced resistance, also were effective in reducing FHB severity, suggesting induced resistance is one mechanism of action. C3 broth cultures also were effective when applied in highly diluted form and when applied 1 week prior to pathogen inoculation. When applied to 8 cultivars of hard red spring wheat in the greenhouse, C3 treatments reduced FHB in 5 cultivars but not in the others. These findings also are consistent with induced resistance. Protection offered by C3 treatments, however, was not systemic and required that C3 be applied uniformly to all susceptible florets. Field tests were conducted in South Dakota and Nebraska to evaluate the efficacy of C3 chitin broth cultures in spring and winter wheat, respectively. In experiments involving two hard red spring wheat cultivars, treatment with C3 reduced FHB severity in ‘Russ’ but not in ‘Ingot’. In three other field experiments comparing C3, the fungicide tebuconazole, and the combination of C3 and tebuconazole, treatments with the bacterial culture alone and the fungicide alone were inconsistent across experiments, each treatment being ineffective in controlling FHB in one experiment. The biocontrol agent–fungicide combination was more consistently effective, reducing FHB incidence or severity in all three experiments. Thus, the potential for using L. enzymogenes C3 as a biological control agent for FHB was demonstrated along with a number of factors that might affect control efficacy in the field.     

Genetic diversity and environmental associations of wild barley,Hordeum spontaneum (Poaceae), in Iran

Genetic diversity and structure of populations of the wild progenitor of barleyHordeum spontaneum in Iran was studied by electrophoretically discernible allozymic variation in proteins encoded by 30 gene loci in 509 individuals representing 13 populations of wild barley. The results indicate that: a)Hordeum spontaneum in Iran is extremely rich genetically but, because of predominant self-pollination, the variation is carried primarily by different homozygotes in the population. Thus, genetic indices of polymorphismP-1% = 0.375, range = 0.267–0.500, and of genetic diversity,He = 0.134, range = 0.069–0.198, are very high. b) Genetic differentiation of populations includes clinal, regional and local patterns, sometimes displaying sharp geographic differentiation over short distances. The average relative differentiation among populations isGst = 0.28, range = 0.02–0.61. c) A substantial portion of the patterns of allozyme variation in the wild gene pool is significanctly correlated with the environment and is predictable ecologically, chiefly by combinations of temperature and humidity variables. d) The natural populations studied, on the average, are more variable than two composite crosses, and more variable than indigenous land races of cultivated barely,Hordeum vulgare, in Iran. — The spatial patterns and environmental correlates and predictors of genetic variation ofH. spontaneum in Iran indicate that genetic variation in wild barley populations is not only rich but also at least partly adaptive. Therefore, a much fuller exploitation of these genetic resources by breeding for disease resistance and economically important agronomic traits is warranted.     

Molecular evidence for recent founder populations and human-mediated migration in the barley scald pathogen Rhynchosporium secalis

Rhynchosporium secalis is an important pathogen of barley globally. Fourteen polymorphic microsatellites were analyzed for 1664 R. secalis isolates sampled from 37 field populations to infer their demographic history. The results falsified the hypothesis that R. secalis co-evolved with its barley host in the Middle East. Populations from Scandinavia had significantly higher allelic diversities, the greatest number of private alleles and the highest genotypic diversities. All but three of the analyzed populations had an excess of gene diversity compared to the number of alleles, consistent with a recent population bottleneck. The remaining populations had a gene diversity deficit consistent with a population expansion following a recent population bottleneck in the last ±100 years. A coalescent analysis revealed that the effective population sizes based on θ, of the analyzed populations were small relative to their ancestral population sizes, indicating that only a fraction of the diversity present in the ancestral populations was transmitted into current populations. These findings are consistent with the hypothesis that the pathogen population on barley experienced a selection bottleneck imposed by the host and/or are founder populations. The mean estimate of migration rates was 2.2 (avg 90% confidence interval = 1.3–3.1). Major migration routes were identified among populations separated by long distances, eg between South Africa and Australia, as well as among North Africa, the Middle East and California, suggesting contemporary exchange of infected barley seed. In contrast with earlier findings, most populations exhibited significant gametic disequilibrium, probably as a result of genetic drift. We conclude that the majority of R. secalis populations have experienced human-mediated migration that led to numerous and relatively recent founder events around the world.     

Effect of inoculum density,nitrogen source and saprophytic fungi on Fusarium wilt of Mexican lime

Summary Mexican lime seedlings were inoculated with 0, 500, 1000, 2000, 4000 and 8000 microconidia ofFusarium oxysporum f. sp.citri per gram of potting media. The percent infection and mean disease severity rating increased with increasing inoculum density of the pathogen. In potting mix infested withAspergillus ochraceus, Penicillium funiculosum andTrichoderma harzianum at 5000 conidia per gram 2 weeks prior to infestation withF. oxysporum f. sp.citri at 0, 1000, 4000, and 8000 microconidia per gram,A. ochraceus reduced,P. funiculosum increased andT. harzianum had no effect on disease severity or pathogen population. OnlyP. funiculosum showed antagonistic activityin vitro against the pathogen. Disease severity and pathogen propagule densitites were greater and pH was lower in potting media fertilized with NH₄–N than in media fertilized with NO₃–N.Portion of M. S. thesis submitted by the senior author to the Graduate School, University of Florida, Gainesville. Florida Agricultural Experiment Stations Journal Series No. 4221.     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

The effect of magnetic field on <Emphasis Type="Italic">Paulownia</Emphasis> tissue cultures

In this study, in vitro tissue cultures of Paulownia tomentosa and Paulownia fortunei were prepared and then exposed to a magnetic flow density of 2.9–4.8 mT and 1 m s⁻¹ flow rate for a period of 0, 2.2, 6.6 and 19.8 s. The magnetic field (MF) increased the regeneration capability of Paulownia cultures and shortened the regeneration time. On the 28th day of culture, the positive effect of magnetic field on plant fresh weight, length, number of leaves and chlorophyll content in node explants of P. tomentosa and P. fortunei was observed. It was found that this effect varied with exposure time. When the cultures were exposed to a magnetic field with strength of 2.9–4.8 mT for 19.8 s, the regenerated P. tomentosa and P. fortunei plants dominated the control plants.     

Microbial pathogens of the coconut pest Oryctes rhinoceros: influence of weather factors on their infectivity and study of their coincidental ecology in Kerala,India

The rhinoceros beetle, Oryctes rhinoceros L., is an economically important pest of the coconut palm. Management of this pest has been accomplished using microbial agents viz., Oryctes virus (OrV) and an entomofungal pathogen Metarhizium anisopliae. Recently an opportunistic bacterial pathogen Pseudomonas alcaligenes has also been noticed to cause septicaemia in the grubs when under stress. To unravel the influence of abiotic weather factors and the interactions amongst these microbial pathogens, a 3 year study was conducted from September 1996 to August 1999 in three of the southern districts of Kerala, India. Of the 6627 grubs and 307 adults collected from various breeding sites of the pest, 5% of the grubs and 22% of the adults had natural virus infection, 3% larvae died of M. anisopliae mycosis and 20% larvae succumbed to bacterial septicaemia. Oryctes virus infection in grubs and adults was negatively correlated to minimum temperature (correlation co-efficient, r = –0.4, and –0.6 respectively, sample size, n = 0). Increase in relative humidity increased the fungal activity (r = 0.8) whereas, maximum temperature had a negative impact (r = –0.7). Occurrence of virus infection in grubs and adults was positively correlated (r = 0.6), supporting the contention of active transmission of the virus pathogen between these two stages. The bacterial septicaemia in the grubs was marginally correlated with virus infection and P. alcaligenes undermined the efficiency of the virus pathogen.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Effect of pH,aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O₂L^–1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L^–1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attainingS. cerevisiae cells suitable for invertase production were: temperature=30°C; pH=5.0; DO=3.3 mg O₂L^–1; (S)=0.5 g L^–1 and sucrose added into the fermenter according to the equations: (V–V_o)=t²/16 or (V–V_o)=(V_f–V_o)·(e^0.6t–1)/10.This work was supported by FAPESP     

Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

Differentiation during asexual reproduction and regeneration in a microturbellarian

Affordable biological technology for the reclamation of wastes and water of the waste streams from intensive livestock units is important in a country short of water. This study tested the concept of reclamation of waste by Streptocephalus macrourus (Crustacea: Anostraca) from the effluent of a high rate algal pond processing livestock wastes. S. macrourus showed a growth efficiency of 39% to 74% when fed optimal rations and cultured at densities between 10 and 400 1^–1. The maximum daily growth rates (0.15–0.21) approximate the growth rates of cladoceran or rotifer cultures managed for maximal biomass production. S. macrourus' ability to withstand crowding enabled the production from S. macrourus cultures (up to 91.8 mg dry mass l^–1 d^–1, or 1241 mg wet mass l^–1 d^–1) to exceed production recorded from cladoceran or rotifer cultures. Temperature influenced growth rate, with the highest growth rate occurring at 24 °C. The dilution rate of continuously fed cultures influenced growth rate, with the optimum dilution rate tested being 10 ml organism ^–1 d^–1. Mass mortality occurred when organisms were held at a density of 4000 l^–1. S. macrourus is able to convert algae grown on livestock waste efficiently into anostracan biomass, and is able to give a very high daily production.     

Biofiltering efficiency in removal of dissolved nutrients by three species of estuarine macroalgae cultivated with sea bass (Dicentrarchus labrax) waste waters 1. Phosphate

The potential of three estuarine macroalgae (Ulvarotundata, Enteromorpa intestinalis andGracilaria gracilis) as biofilters for phosphate ineffluents of a sea bass (Dicentrarchus labrax) cultivationtank was studied. These seaweeds thrive in Cádiz Bay and were alsoselected because of their economic potential, so that environmental andeconomicadvantages may be achieved by future integrated aquaculture practices in thelocal fish farms. The study was designed to investigate the functioning of Pnutrition of the selected species. Maximum velocity of phosphate uptake (2.86mol PO₄ g^–1 dry wth^–1) was found in U. rotundata.This species also showed the highest affinity for this nutrient. At low flowrates (< 2 volumes d^–1), the three species efficientlyfiltered the phosphate dissolved in the waste water, with a minimum efficiencyof 60.7% in U. rotundata. Net phosphate uptake rate wassignificantly affected by the water flow, being greatest at the highest rateassayed (2 volumes d^–1). The marked decrease in tissue P shownby the three species during a flow-through experiment suggested that growth wasP limited. However, due to the increase in biomass, total P biomass increasedinthe cultures. A significant correlation was found between growth rates and thenet P biomass gained in the cultures. A three-stage design under low water flow(0.5 volumes d^–1) showed that the highest growth rates (up to0.14 d^–1) and integrated phosphate uptake rates(up to 5.8 mol PO₄ ^3– g^–1dry wt d^–1) were found in E.intestinalis in the first stage, with decreasing rates in thefollowing ones. As a result, phosphate become limiting and low increments oreven losses of total P biomass in these stages were found suggesting thatphosphate was excreted from the algae. The results show the potential abilityofthe three species to reduce substantially, at low water flow, the phosphateconcentration in waste waters from a D. labrax cultivationtank, and thus the quality of effluents from intensive aquaculture practices.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.