Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in controlling Anopheles arabiensis and Culex quinquefasciatus mosquitoes

The present study aimed to investigate, the larvicidal, adult emergence inhibition and oviposition deterrent activity of aqueous leaves extract of Calotropis procera against Anopheles arabiensis and Culex quinquefasciatus as natural mosquito larvicide. The larvicidal activity was monitored against 2nd, 3rd and 4th instar larvae of each mosquito species 24 h post-treatment. Adult emergence inhibition activity was tested by exposing 3rd instar larvae of each mosquito species to different concentrations of extracts (200, 400, 600, 800 and 1000 ppm for An. arabiensis and 100, 200, 300, 400, 500 and 600 ppm for Cx. quinquefasciatus). Probit analysis was used to analyze data from bioassay experiments. The oviposition deterrent activity was tested by using three different concentrations of extracts (1000, 500 and 200 for An. arabiensis, and 1000, 500 and 100 for Cx. quinquefasciatus) that caused high, moderate and low larval mortality in the larvicidal experiment against 3rd instar larvae. It was found that, LC50–LC90 values calculated were 273.53–783.43, 366.44–1018.59 and 454.99–1224.62 ppm for 2nd, 3rd and 4th larval instars, respectively, of An. arabiensis and 187.93–433.51, 218.27–538.27 and 264.85–769.13 ppm for 2nd, 3rd and 4th larval instars, respectively, of Cx. quinquefasciatus. Fifty percent of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively. The pupal stage was not affected till a concentration of 5000 ppm. The extract showed oviposition deterrence and effective repellence against both mosquito species at different concentrations, with the observation on that maximal eggs were laid in low concentration of extract. These results suggest that the leaves extract of C. procera possess remarkable larvicidal, adult emergence inhibitor, repellent and oviposition deterrent effect against both An. arabiensis and Cx. quinquefasciatus, and might be used as natural biocides for mosquito control.     

Nitrogen limitations to field bean productivity: A comparison of combined nitrogen applications with Rhizobium inoculation

Summary Nitrogen limitations to the yield of a field crop ofVicia faba have been examined. Application of nitrogen totalling 560 kg/ha increased dry matter yield at flowering by 674 kg/ha (32%) and grain yield at final harvest by 1.6 tonnes/ha (24%). Attempts to reduce nitrogen limitations by replacing the native rhizobia with strains ofRhizobium leguminosarum selected for high rates of nitrogen fixation were unsuccessful but the introduction of poor rhizobia reduced grain yield. The reasons for this and the implications of the results for crop improvement are discussed.     

Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa

The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared. In the laboratory, both species ingested a similar amount of B. sphaericus spores when fed c. 2 x 10(5) spores per ml for 30 min. However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h. In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B. sphaericus 2362, containing 2 x 10(10) spores/g. It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B. sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B. sphaericus.     

High-latitude<Emphasis Type="Italic"> Acropora cervicornis</Emphasis> thickets off Fort Lauderdale,Florida, USA

Baseline studies conducted in 1998 document the presence of robust, non-reef-building Acropora cervicornis thickets in shallow (3–7 m depth), near-shore waters off the coast of Fort Lauderdale, Florida, USA. These thickets thrive in a high-latitude environment, the northernmost in the continental USA, in the midst of potential anthropogenic stressors. Within thickets, the spatial variation in mean percent coral cover, macroalgal cover, scleractinian species richness, density of A. cervicornis juveniles, and the density and size of A. cervicornis colonies and fragments were recorded. Thicket size ranged between ~0.1 and 0.8 ha and mean coral cover varied between ~5 and 28%, with A. cervicornis accounting for ~87–97% of all scleractinians. Mean A. cervicornis colony and fragment densities per thicket were 1.3–3.3 colonies m^–2 and 0.7–2.8 fragments m^–2, respectively. Recruit densities varied between 0 and 1 ind. m^–2. White band disease was detected at all thickets, with a mean A. cervicornis colony surface area affected of 1.8%. For all thickets, densities of the corallivorous polychaete Hermodice carunculata ranged between ~18 and 86 ind. ha^–1, with predation scars affecting <0.2% of the A. cervicornis cover. These flourishing A. cervicornis thickets off Fort Lauderdale provide an interesting counterpoint to the declining and disease-stricken A. cervicornis populations reported in the Florida Keys and wider Caribbean.     

UV protectants for the biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary toxins from UV radiation

The UV protectant properties of 26 natural and synthetic compounds were investigated for a biopesticide based on an indigenously isolated strain (ISPC-8) of Bacillus sphaericus Neide. In initial screening, spores of ISPC-8 with 0.1% (w/w for solid and v/w for liquid materials) concentration of different compounds were exposed to UV-B radiation (4.9 × 10⁵ J/m²) for 6 h and their spore viability and larvicidal activity were studied. The larvicidal activity was evaluated against third-instar larvae of Culex quinquefasciatus Say. There was a complete loss of spore viability (1.4% viable spores) and partial reduction in larvicidal activity (57.7% of original activity) after exposure of spores to UV-B for 6 h. However, spore viability as well as larvicidal activity protected significantly when spores were mixed with different compounds before exposing them to UV-B. Among the different compounds tested benzaldehyde, congo red, para-aminobenzoic acid (PABA) and cinnamaldehyde were found to be promising in protecting the spores from UV-B radiation. The presence of binary toxins (41.9 kDa and 51.4 kDa) in protected and unprotected samples were examined by SDS–PAGE. The binary toxin bands disappeared in unprotected spores after 24 h of exposure to UV-B, whereas toxin bands were distinctly visible when spores with benzaldehyde and cinnamaldehyde were exposed to UV-B for 96 h and 120 h, respectively. Congo red and PABA were found to be most effective in protecting binary toxins even after 168 h of exposure to UV-B. Incorporation of these promising UV protectant compounds in biopesticides would help in protecting the spores from the adverse effects of UV radiation and prolong the persistence of biopesticides under field conditions.     

Microplate assay of elevated esterase activity in individual pyrethroid-resistant mosquitoes

Involvement of esterase-mediated hydrolysis as a mechanism of pyrethroidresistance in three species of mosquitoes,viz., Aedes aegypti, Culex quinquefasciatus andAnopheles Stephensi was investigated by microplate assay of Β-esterases in individual larva and adult female and male mosquitoes. Assuming an absorbance value of 0.4 and above at 555 nm as the threshold level of elevated esterase activity which confers resistance, frequency distributions of such individual test mosquitoes were constructed in resistant and susceptible populations. The results indicate the involvement of ester hydrolysis of Pyrethroids as a predominant mechanism of pyrethroid-resistance in the larvae ofCulex quinquefasciatus but not inAedes aegypti. However, a marginal role of esterases is indicated in the larvae ofAnopheles stephensi.     

Oviposition preference and offspring performance in container breeding mosquitoes: evaluating the effects of organic compounds and laboratory colonisation

1. The preference–performance hypothesis (PPH) predicts that organisms lacking parental care should oviposit in habitats that optimise offspring performance. Preference–performance relationships were investigated for the Asian tiger mosquito (Aedes albopictus Skuse) and the southern house mosquito (Culex quinquefasciatus Say) (Diptera: Culicidae), two medically important container‐breeding species, in response to an organic chemical blend mimicking decaying plant matter. Additionally, the effects of long‐term laboratory colonisation of Cx. quinquefasciatus using wild and laboratory strains were evaluated. 2. Oviposition bioassays were conducted by releasing gravid mosquitoes into field enclosures with automobile tires containing low and high concentrations of the chemical blend, and water controls. The offspring were then reared in water collected from the tires in which they were deposited. 3. Aedes albopictus and wild Cx. quinquefasciatus laid more eggs in the chemical blend than water controls but did not differentiate between the low and high concentrations. Conversely, laboratory Cx. quinquefasciatus only preferred the high concentration to the low concentration. No statistical associations between oviposition preference and larval survival were found, as the chemical blend did not affect survivorship of either species. 4. The oviposition preference for the chemical blend over water controls suggests that both species oviposit in the best available resource environment, but further studies are needed before conclusions regarding preference–performance relationships can be drawn. 5. It was found that long‐term laboratory colonisation affects the oviposition behaviour in Cx. quinquefasciatus, suggesting that behavioural studies on laboratory strains are not always applicable to wild populations.     

Soil and litter respiration rates in different microhabitats of a mixed oak-conifer forest and their control by edaphic conditions and substrate quality

Summary Seven isolates belonged toA. brasilense and 3 belonged toA. lipoferum. Isolates having more denitrifying capacity fixed less nitrogen in nitrogen free semi-solid malate medium. One strain ofA. lipoferum having high nitrogen fixing capacity with negative test for denitrification was tested as inoculant to supplement the nitrogen need of a wheat crop in field condition with different doses of N with and without the inoculant. While control without nitrogen yielded 1260 kg/ha the yield in inoculated treatment was 2070 kg/ha resulting in significant increase. In a treatment receiving 40 kg N/ha the grain yield was 2370 kg/ha as against yield of 3110 kg/ha in a similar treatment receiving fertiliser plus inoculant. Thus increase in yield was about 30%. Further the treatment receiving 80 kg N/ha yielded 2970 kg/ha as against yield of 4150 kg/ha in a treatment receiving inoculant alongwith the above dose of the fertiliser. Thus increase in yield due to application of inoculant was about 36%. Similarly, the uptake of N in different treatments was augmented due to inoculation of seeds with the culture.     

Effects of a granulosis virus,andBacillus thuringiensis on life-table parameters of the potato tubermoth,Phthorimaea operculella

Experiments were conducted to evaluate the effect of 3 different concentrations of a granulosis virus (GV), and a standard application ofBacillus thuringiensis Berliner, on the life table parameters of the potato tubermothPhthorimaea operculella (Zeller) in the laboratory. Survival from egg to adult emergence was lowest (0.4%) and no reproduction occurred when larvae were fed on tubers treated withB. thuringiensis (i.e. 200 mg Thiricide/kg potatoes). Survival was 0.8, and 11.1% on tubers treated with 2.0, and 0.2 larval equivalents/kg potatoes (LE) of the GV, respectively, and was significantly (p<0.05) reduced compared to that for the lowest GV concentration (0.002 LE), and the control, i.e. 34.7, and 32.5% survival, respectively. However, generation time was not affected by the GV and fecundity was reduced only at the highest concentration. As a result, the intrinsic rate of increase (r_m) was significantly reduced at the highest, slightly reduced at the intermediate, and was not different for the lowest GV concentration as compared to the control, i.e. 0.0004, 0.0071, and 0.0100 compared to 0.0105, respectively. The implications for a potential biological control of the potato tubermoth in rustic potato stores are discussed.      

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×10⁹ c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L₄ Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.The authors are at the Department of Applied Microbiology & Brewing, Anambra State University of Technology, P.M.B. 5025, Awka, Nigeria.     

Resistance to theBacillus sphaericus toxin in cultured mosquito cells

Summary Increasing doses ofBacillus sphaericus toxin were used to select a toxin-resistant cell line from theCulex quinquefasciatus line. This resistant cell line was proven to beC. quinquefasciatus in origin by isozyme analysis and karyotype. The resistant line bound fluorescent-labeled toxin as did the unselected susceptible line. A high level of resistance was quickly achieved, and this level was maintained after 4 mo. culture in the absence of toxin. This work was supported by grant number RO1 AI 22702 from the National Institutes of Health, Bethesda, MD.     

Complexity of species conservation in managed habitats: interaction betweenMaculinea butterflies and their ant hosts

Europe's five species ofMaculinea butterfly are examples of endangered species adapted to live in traditional, cultural landscapes. All are threatened with extinction in Western Europe because of recent changes in land use. This is illustrated by an historical account of the extinction of the BritishMaculinea arion populations, despite many conservation attempts. It is shown how the failures proved to be due to ignorance of the key factor forM. arion, its specialization on a single ant host,Myrmica sabuleti. A brief account is given of research that shows how each of the five species is similarly dependent upon a separate hostMyrmica ant species and how each has an interesting and rare specific parasitoid. Steps for the practical conservation of existingMaculinea populations including the obligation, under the Bern Convention, to re-establish nationally extinct species are outlined. The procedure and problems involved in re-establishment are illustrated with reference to the successful programme forM. arion in Britain. The best way of ensuring robust populations ofMaculinea butterflies is to manage habitats to optimize the density and distribution of the required species ofMyrmica host and, secondarily, the distribution of the larval food plant. The value of single species conservation in cultural habitats is discussed. It is concluded that this is possible to achieve and that other rare organisms also often benefit, but only when conservation measures are based on the results of detailed autecological research.     

Evaluation of the availability ofAzolla-N and urea-N to rice using15N

Summary The symbiotic association of the water fernAzolla with the blue-green algaAnabaena azollae can fix 30–60 kg N ha^–1 per rice cropping season. The value of this fixed N for rice production, however, is only realized once the N is released from theAzolla biomass and taken up by the rice plants. The availability of N applied asAzolla or as urea was measured in field experiments by two¹⁵N methods. In the first,Azolla caroliniana (Willd.) was labelled with¹⁵N in nutrient solution and incorporated into the soil at a rate of 144 kg N ha^–1. The recovery ofAzolla-N in the above ground parts of rice [Oryza sativa (L) cv. Nucleoryza] was found to be 32% vs. 26% for urea applied at a rate of 100 kg N/ha; there was no significant difference in recovery. In the second, 100 kg N/ha of¹⁵N-urea was applied separately or in combination with either 250 or 330 kg N ha^–1 of unlabelledAzolla. At the higher rate, the recovery ofAzolla-N was significantly greater than that of urea. There was a significant interaction when both N sources were applied together, which resulted in a greater recovery of N from each source in comparison to that source applied separately. Increasing the combined urea andAzolla application rate from 350 kg N ha^–1 to 430 kg N ha^–1 increased the N yield but had no effect on the dry matter yield of rice plants. The additional N taken up at the higher level of N application accumulated to a greater extent in the straw compared to the panicles. Since no assumptions need to be made about the contribution of soil N in the method using¹⁵N-labelledAzolla, this method is preferable to the¹⁵N dilution technique for assessing the availability ofAzolla-N to rice. Pot trials usingAzolla stored at –20°C or following oven-drying showed that both treatments decreased the recovery of N by one third in comparison to freshAzolla.     

Effects of wastewater sludge and woodchip combinations on soil properties and growth of planted hardwood trees and willows on a restored site

Sludge from wastewater treatment plants and woodchips produced from urban tree pruning residues were used to improve soil conditions of a degraded site restored by planting trees and shrubs. The release of soil nitrogen resources was set by the proportions of sludge and woodchips applied. Combining 187 kg N/ha of sludge in one application with 200 m³/ha of woodchips instead of 100 m³/ha reduced first year N mineralization by 50%. The same can be said for the application of 125 kg N/ha of sludge in two applications, over 2 years. The degradation of sludge–woodchips and nitrate leaching was reduced even with the smallest sludge application. Survival and growth of Acer saccharinum L., Fraxinus pennsylvanica Marsh. and Salix discolor Muhl. did not vary much with sludge and woodchips quantities. However, differential soil nitrogen availability and the degradation process of organic amendments induced by the treatments may result in better sustainable growth for planted woody species. Some undesirable elements were measured in the soil, particularly Fe, Cd, Pb and NO₃–N. Because of these, care should be taken when choosing sites to be restored using sludge and woodchips.     

The effect of stem and cob borers on maize subjected to different nitrogen treatments

The influence of four nitrogen levels (0, 60, 90 and 120 kg N/ha) on growth of maize and development of lepidopterous pests was investigatdd in a field trial. Nitrogen had a positive effect on both plant growth variables (plant height, stem diameter and yield), and development and survival ofSesamia calamistis andEldana saccharina, and thereby increased the incidence of dead hearts and stem tunneling. However, the percent yield loss due to artificial infestation decreased with increasing N application rate from 20% to 11% in the in the 0kg/ha and 120kg/ha treatment, respectively. Using a multiple regression analysis, plant height, plant diameter and stem tunneling were found to be the most important variables explaining the variability in maize yield.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Suppression ofOstrinia nubilalis byTrichogramma nubilale in sweet corn

We conducted inundative release experiments withTrichogramma nubilale (Hymenoptera: Trichogrammatidae) to suppressOstrinia nubilalis (Lepidoptera: Pyralidae) in sweet corn (Zea mays): two experiment duringO. nubilalis first generation and three experiments during second generation. Five measurements of ear and stalk damage were used to assess.O. nubilalis control in treated and untreated plots within each experimental field. In one experiment during second generation, naturalO. nubilalis populations were sufficiently high to demonstrate that the parasitoids (three releases totaling 4.4 million parasitoids per ha) parasitized an estimated 57.4% of the placedO. nubilalis egg masses and reduced the mean number ofO. nubilalis larvae per ear by 97.4% the number of tunnels per stalk by 92.9%, and the number of larvae per stalk by 94.3% in the release plot. Ear damage in this experiment was suppressed to meet acceptable standards for use in cut-corn commercial processing. Larval mortality was apparently density independent, which implies that density-dependent larval loss would not compensate for egg parasitism byT. nubilale.     

The effect of oxygen tension on the production of Bacillus thuringiensis subsp. israelensis toxin active against Aedes aegypti larvae

An optimized batch production of Bacillus thuringiensis subsp. israelensis was made in a stirred Bioflo III reactor using a previously selected medium, and operating conditions in the range of 100–500 rev/min stirrer speeds and 0.2–1 air flow/culture medium volume/minute (v/v/m) aeration rates, including five combinations; at the end of fermentation, dry powders were recovered and evaluated against Aedes aegypti larvae at 0.05 mg/l. Later, the lethal concentration inducing 50% mortality (LC₅₀) was determined for the two most toxic powders. The bioinsecticide yields varied from 9.1 to 15.7 g/l and the total fermentation times ranged between 18 and 30.3 h. The toxicity varied for two powders much more than for the others. These were for combinations with 300 rev/min:1 v/v/m and 500 rev/min:0.6 v/v/m, giving mortality percentages of 47.2 and 59.7, with LC₅₀ values of 0.2675 and 0.0685 mg/l, respectively. A t test showed no significant difference. However, the larvicidal powder produced with 300 rev/min:1 v/v/m gave more variable toxicity than the powder obtained with 500 rev/min:0.6 v/v/m.