Decreased incidence of disease caused by Sclerotinia sclerotiorum and improved plant vigor of oilseed rape with Bacillus subtilis Tu-100

Sclerotinia sclerotiorum causes serious yield losses in oilseed crops worldwide. Bacillus subtilis Tu-100 significantly reduced (P≤0.05) the incidence of disease caused by S. sclerotiorum on oilseed rape at harvest in two trials conducted in fields artificially infested with this pathogen. Mean plant dry weight was significantly greater (P≤0.05) and mean plant length was significantly greater (P≤0.07) at the seven-true-leaf stage with the Tu-100 treatment than with the control. Mean seed yield per 120 plants at harvest was significantly greater (P≤0.05) in the second field trial with treatments containing isolate Tu-100. B. subtilis Tu-100 also promoted the growth of hydroponically grown oilseed rape. Plants were approximately 15% greater in dry weight (P≤0.0001) and 6% greater in length (P≤0.0025) when grown in the presence of isolate Tu-100 in Hoagland’s solution, compared with the noninoculated control. In gnotobiotic studies, the lacZ-tagged strain B. subtilis Tu-100(pUC18) was detected within all roots of oilseed rape. Isolate Tu-100 did not persist in the ectorhizosphere of oilseed rape. Populations of this isolate decreased from 8.5×10⁸ colony-forming units (CFU) per seed to approximately 10² CFU in the plant ectorhizosphere within 30 days of sowing in autoclaved soil.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Microencapsulation of Bacillus subtilis B99-2 and its biocontrol efficiency against Rhizoctonia solani in tomato

Biological control is a promising approach to protecting plants from disease. Bacillus subtilis has been widely used in agriculture for promoting plant growth and biocontrol. However, their short shelf life limits the application of biological pesticides. The objectives of this study were to develop a microencapsulation procedure of B. subtilis B99-2 using maltodextrin and gum arabic as wall materials to determine the optimum conditions of spray-drying in microencapsulation, evaluate storage stability of microcapsules, and assess their biocontrol efficiency against Rhizoctonia solani in tomato under field conditions. We microencapsulated the Bacillus thallus by spray-drying with various concentrations of the wall material. Maltodextrin was found to be an efficient wall material, especially at concentrations higher than 80%, while gum arabic did not affect the bacterial survival rate. The mean survival rate of B. subtilis was more than 90%, when spray drying was performed at 145 °C, with a feed flow rate of 550 mL h⁻¹, and a spray pressure of 0.15 MPa. B. subtilis microcapsule survival rate was 87.53% after 540 d of storage, which was a longer shelf life than that of wettable powders. Moreover, its biocontrol efficacy reached 79.91% when a dosage of 300 g hm⁻² was used, the microcapsule showed higher control efficacy than Thiram wettable powder against R. solani in tomato under field conditions. All these characteristics indicated that B. subtilis microcapsules have the potential to become a successful biocontrol product.     

Anthraquinone-based bird repellent for sunflower crops

Non-lethal alternatives are needed to manage bird damage to confectionery and oilseed sunflower crops (Helianthus annuus). Ring-necked pheasants (Phasianus colchicus) can cause localized damage to newly planted sunflower, and blackbirds (Icterids) damage ripening sunflower annually in the United States of America. We conducted seed germination experiments, a repellent efficacy study with ring-necked pheasants and Avipel^® repellent (a.i. 50% 9,10-anthraquinone), and laboratory and field efficacy studies with common grackles (Quiscalus quiscula) and Avipel^®-treated confectionery sunflower. Compared to the germination of seeds not treated with anthraquinone, we observed no negative effects of up to 12,223 ppm, 14,104 ppm, and 11,569 ppm anthraquinone seed treatments for germination of confectionery sunflower, oilseed sunflower, and canola seeds, respectively. Pheasants avoided emergent sunflower seedlings (12 days post-planting) from 15,800 ppm anthraquinone seed treatments during a caged preference test (P = 0.045). We observed a positive concentration–response relationship (P = 0.001) and predicted a threshold concentration (i.e., 80% repellency) of 9200 ppm anthraquinone for common grackles offered Avipel^®-treated confectionery sunflower seeds. Grackles also reliably discriminated between untreated sunflower and seeds treated with 1300 ppm anthraquinone in captivity (P < 0.001). During our field efficacy study for ripening confectionery sunflower, we observed 18% damage among anthraquinone-treated enclosures and 64% damage among untreated enclosures populated with common grackles (P < 0.001). Harvested seed mass averaged 2.54 kg (dry weight) among treated enclosures and 1.24 kg among untreated enclosures (P < 0.001). Our laboratory and field efficacy data provide a reliable basis for planning future field applications of anthraquinone-based repellents for protection of sunflower crops. Supplemental field efficacy studies are necessary for development of an effective avian repellent and management of avian depredation of ripening agricultural crops, including oilseed sunflower.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Management of white mold in processing tomatoes by Trichoderma spp. and chemical fungicides applied by drip irrigation

Field trials were carried out to evaluate six treatments combining biological agents and chemical fungicides applied via chemigation against white mold (Sclerotinia sclerotiorum) on processing tomatoes. The experiment was performed in Goiânia, Brazil, with tomato hybrid Heinz 7155 in 2009 and 2010 in a field previously infested with S. sclerotiorum sclerotia. Treatments were arranged in a randomized complete block design in a 2 × 3 factorial structure (with and without Trichoderma spp. 1.0 × 10⁹ viable conidia mL⁻¹ ha⁻¹) × fluazinam (1.0 L ha⁻¹), procimidone (1.5 L ha⁻¹) and control, applied by drip irrigation. Treatments were applied three times 10 days apart, starting one month after transplanting. Each treatment consisted of plots with three 72-meter rows with four plants m⁻¹ and 1.5 m spacing between rows, with three replications. Based on disease incidence evaluated weekly, the area under the disease progress curve (AUDPC) was obtained. Yield and its components were evaluated in addition to fruit pH and °Brix. Results were subjected to ANOVA, Scott-Knott (5%), and regression analysis. Biocontrol using Trichoderma spp. via chemigation singly or in combination with synthetic fungicides fluazinam and procimidone reduced AUDPC and increased fruit yield up to 25 t ha⁻¹. The best treatment for controlling white mold also increased pulp yield around 1.0 and 7.0 t ha⁻¹ in 2009 and 2010, respectively. The present work demonstrated the advantages of white mold biological control in processing tomato crops, where drip irrigation favored Trichoderma spp. delivery close to the plants and to the inoculum source.     

The seed bank in a subtropical freshwater marsh: implications for wetland restoration

Seed bank samples were collected from Huli Marsh, a subtropical shallow water mountainous marsh in Hunan Province, South China. Core samples were divided into upper and lower layers (each 5 cm in depth) and allowed to germinate in three water levels (0, 5 and 10 cm) over a 4-month period. A total of 51 species germinated and the mean density was 9211 ± 7188 seedlings m⁻². In the top 5 cm 41 species and 5747 ± 5111 seedlings m⁻² germinated, whereas 40 species and 3464 ± 3363 seedlings m⁻² did so from 5–10 cm. Germinated seedling density was significantly higher in the upper layer, largely due to differences in eight species. With increasing experimental water depth, less seedlings germinated: respectively, 9788 ± 7157 m⁻², 2050 ± 2412 m⁻² and 1978 ± 2616 m⁻², of 44, 21 and 19 species, submerged under 0, 5 or 10 cm. Seven species could emerge only in 0 water level. Vallisneria natans occurred only in 5 cm water, whereas Ottelia alismoides occurred in 10 cm water. In the vegetation survey of the marsh, 25 species were recorded, which was less than half of the species recorded in the seed bank. The top 10 dominants in the standing vegetation, accounting for 89% of vegetation abundance, represented only 10% in the seed bank. Twenty germinated species that also occurred in the standing vegetation accounted for 56% of the total seed bank. Our observed number of species germinating from a Chinese wetland seed bank is within the range observed elsewhere in the northern hemisphere (15–113 species).     

Successful storage of protocorm-like bodies of hybrid Cymbidium (Orchidaceae) under low temperature conditions

Low temperatures result in lower metabolic cellular activity, thus slowing down cell division and growth. This is advantageous where a plant scientist might seek to store important germplasm without the risks associated with low temperature storage. In this study, two cold temperatures above freezing, namely 4 and 10 °C, were tested to assess for how long PLBs could be preserved without a significant loss in regeneration ability (i.e., the ability to form neo-PLBs). Control treatments were cultured at 25 °C on Teixeira Cymbidium (TC) medium at a 16-h photoperiod at a photosynthetic photon flux density (PPFD) of 45 μmol m⁻² s⁻¹. For the cold treatments, each was replicated in the dark and at low light intensity (12-h photoperiod and a PPFD of 10 μmol m⁻² s⁻¹). All cultures were sub-cultured six times onto fresh medium every 60 days, for approximately 1 year. On the 7th subculture, all neo-PLBs were prepared uniformly and replated onto standard TC medium under light conditions described above for the control. 45 days after the 7th subculture and just before subcultures 1–6, the number of neo-PLBs per half-PLB was measured. The number of neo-PLBs that formed under different treatments depended strongly on the temperature and light conditions with most neo-PLBs forming under control conditions, although that number dropped significantly as the temperature was dropped to 10 °C and then even more to 4 °C, the same trend being observed when explants were cultured and subcultured under dim light, with organogenesis being more strongly negatively influenced in darkness. For all low-temperature treatments, as well as the dimmed light and darkness treatments, the number of neo-PLBs increased significantly when recultured, on the 7th subculture, onto control TC medium under control environmental conditions, almost as high as the control values. In contrast, the control values decreased, with significantly fewer neo-PLBs by the 7th subculture relative to the control, indicating that new PLBs should be induced from shoot cultures at least once a year to maintain their vitality.     

RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 × 10⁹ cfu μg^− 1 ml^− 1 with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 × 10⁶ and 1 × 10⁸ cfu μg^− 1 ml^− 1. The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10³ times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10⁶ times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

Evaluation of biological seed treatments in combination with management practices for the control of Fusarium dry rot of potato

Seed-borne diseases of potato represent a significant constraint to potato production in the US. The use of an effective fungicide in combination with good management practices during cutting and storage, prior to planting, is essential to reducing disease. The efficacy of two biocontrol agents (Bacillus subtilis and Trichoderma harzianum), and a commercially formulated mixture of the chemicals fludioxonil plus mancozeb, applied as seed treatments in combination with different management practices, were evaluated over two years for the control of seed piece decay and sprout rot caused by Fusarium sambucinum. Treatments were made 10 days prior to planting and at planting, and tubers were re-stored at either 18 °C and 95% RH with forced air ventilation at 5950 l min⁻¹ (optimal conditions), at 25 °C in the dark without ventilation (sub-optimal), or not stored at all prior to planting. Seed piece and sprout health were evaluated in vitro and agronomic impacts evaluated in field experiments. Results showed that the biological control agents B. subtilis and T. harzianum provided good control of sprout rot and seed piece decay caused by F. sambucinum, when seed was re-stored under optimal conditions or not re-stored at all. Under optimal conditions, treatment with B. subtilis reduced sprout rot and seed piece decay on average by 66% and 84%, respectively. Treatment with T. harzianum reduced sprout rot and seed piece decay on average by 70% and 81%, respectively. Treatment with fludioxonil + mancozeb reduced sprout rot and seed piece decay under both re-storage regimes. Under optimal conditions, disease incidence and severity was reduced on average by 81% and 97%, respectively. Neither biological control agent reduced seed piece decay incidence under either re-storage regime compared to the untreated controls.     

An ozone response relationship for four Phleum pratense genotypes based on modelling of the phytotoxic ozone dose (POD)

In the last decade extensive research has focused on the development of dose–response relationships based on stomatal plant ozone uptake (phytotoxic ozone dose, POD). So far most work has concentrated on crops and forest trees. This study provides a flux-based dose–response function for timothy (Phleum pratense), a widespread grassland species, which can be used in risk assessment for ground-level ozone. In 1996 and 2001 timothy was exposed in open-top chambers to ozone concentrations ranging from around 10 nmol mol⁻¹ in the charcoal filtered treatments up to 60 nmol mol⁻¹ in the fumigated treatments (08:00–20:00) in. In 1996 there was a negative effect of ozone on biomass production in the non-filtered treatment while in 2001 no such ozone effect in the non-filtered treatment could be seen. Measurements of stomatal conductance on four timothy genotypes in 2001 were used to calibrate a Jarvis-type multiplicative stomatal conductance model. The maximum conductance varied between the genotypes, from 477 to 589 mmol O₃ m⁻² s⁻¹ (projected leaf area). The model includes functions describing the reduction of stomatal conductance of senescing leaves and the direct effects on stomatal conductance by light, temperature and water vapour pressure deficit. A function describing ozone induced senescence of the leaves was included since exposure to ozone is known to cause premature senescence. The function for ozone was applied when it suggested ozone to be more limiting to stomatal conductance than phenology. To avoid overestimation of stomatal conductance in days with high VPD, a function reflecting the effect on leaf water potential on stomatal conductance was included. Comparison between modelled and measured conductance for the four timothy genotypes resulted in an r² value at 0.57 and a very small average deviation of observed from modelled values. The calibrated stomatal conductance model was used to estimate the accumulated POD, i.e. the accumulated stomatal flux of ozone, of the plants in the 1996 and 2001 experiments. The strongest relationship between ozone relative effects on biomass was obtained when POD was accumulated from 105 degree days after emergence to 1000 degree days after emergence, and integrated using an uptake rate threshold of 7 nmol m⁻² s⁻¹ (POD₇). The response relationship between biomass and POD₇ resulted in an r² value of 0.71 over all four genotypes. This r² value was somewhat higher than for the corresponding relationship based on the accumulated ozone exposure over 40 nmol mol⁻¹ (AOT40; r² = 0.66). With an uptake rate threshold at 7 nmol m⁻² s⁻¹, ozone concentrations above ∼20 nmol mol⁻¹, contribute to reduce the biomass production of timothy if meteorological conditions promote maximum stomatal conductance.     

Effects of pellet morphology on broth rheology in fermentations of Aspergillus terreus

Effects of pellet morphology on broth rheology are reported for pelleted submerged cultures of the lovastatin producing filamentous fungus Aspergillus terreus, growing in fluidized bed and stirred tank bioreactors. The pellet diameter and compactness were affected by the agitation intensity of the broth; however, the total biomass productivity was not affected. In fluidized beds and stirred tanks with agitation intensity of up to 300 rpm (impeller tip speed of 1.02 m s⁻¹), the fungal pellets were stable at diameters of up to about 2300 μm. In more intensely agitated stirred tanks (≥600 rpm; impeller tip speed of ≥2.03 m s⁻¹), the stable pellet size was only about ≤900 μm. The biomass concentration and the pellet diameter were the main factors that influenced the flow index and the consistency index of the power-law broths. Because the biomass productivity was the same in all experiments in a given type of reactor and the oxygen concentration was kept at ∼400% of air saturation, the pellet size and morphology were not influenced by oxygen mass transfer effects. Pellets were always dense in the core region and no necrosis of the biomass occurred.     

Questions and answers to edibility problem of the Canavalia ensiformis seeds – A review

The seed of Canavalia ensiformis (jackbean), a highly productive large-seeded tropical legume, contains about 300 g crude protein and 600 g carbohydrates kg⁻¹ dry matter. It, however, contains toxic and antinutritional factors which limit its use as human food or animal feed. The trypsin inhibitors in the seed have, however, been reported to be easily inactivated by moist heat. The toxic alkaline non-protein amino acid, canavanine (present at about 50 g kg⁻¹ seed dry matter), a naturally occuring analog of l-arginine, has also been reported to induce reduced feed intake in non-ruminants but at the equivalent of about 300 g kg⁻¹ dietary level of the raw seed, or in the condition of dietary arginine deficiency. The concentrations of the specialized secondary plant biomolecules – cyanogenic glycosides, saponins, alkaloids and terpenoids – have also been shown to drop below detectable levels following 1 h of cooking. Urease and canatoxin which were isolated from the seed are highly toxic if injected into experimental animals but exert no toxic effects if orally administered, and thus cannot be classified as antinutritional factors. Concanavalin A (Con A), the most studied of plant lectins, appears to be the most important toxic and antinutritional factor in the seed, being highly resistant to heat treatments and to proteolytic digestion in the gut. It requires 3 h of cooking at 96°C or 45 min of pressure cooking; 48 h of soaking the seed in water prior to cooking for 2 h; 72 h of soaking in water prior to ordinary cooking for 1 h; pressure-cooking for 15 min, to completely eliminate it from the seed. Complete inactivation of Con A in the seed can, however, be easily achieved if the seed is broken into pieces and cooked for 1 h or pressure-cooked for 15 min.     

Vase life of cut rose cultivars ‘Avalanche’ and ‘Fiesta’ as affected by Nano-Silver and S-carvone treatments

Rosa hybrida L. is an important commercial cut flower. The vase life of this flower is usually short due to vascular occlusion. We assessed the effect of Nano-Silver (NS) and S-carvone in prolonging the vase life of R. hybrida L. cv. ‘Avalanche’ and ‘Fiesta’. Hence, an experiment involving the treatment with NS at 0, 50, 100, and 200 mgL^− 1 and S-carvone at 0, 0.25, 0.5, and 0.75 mgL^− 1 with 10 replicates was conducted. Applying NS pulse treatments increased vase life, water uptake rate, and fresh weight and reduced the number of bacteria, water loss, stomatal conductance and transpiration rate. However, S-carvone treatments did not have a positive effect on the vase-life parameters of cut rose flowers. NS pulse treatments increased relative fresh weight (RFW), and water uptake rate (WUR) and decreased water loss (WL) (%) by 10, 89 and 31% for cultivar ‘Avalanche’, compared to the controls, respectively. Application of 200 mgL^− 1 NS led to the highest vase life (18 days) for roses. The results show that NS increased vase life by suppressing stomatal opening, decreasing transpiration from leaves and inhibiting bacterial proliferation.     

Insecticidal properties of Pimpinella anisum essential oils against the Culex quinquefasciatus and the non-target organism Daphnia magna

The efficacy of an essential oil obtained from Pimpinella anisum fruits and its major compound, trans-Anethole, was tested on the eggs, larvae and adults of Culex quinquefasciatus. While causing no significant mortality on eggs, other tested stages were very sensitive to the essential oil and trans-Anethole. LC₅₀ for the 2nd to 4th instar larvae was estimated as 26–27 μL·L^− 1 and 15–19 μL·L^− 1 for the essential oil and trans-Anethole, respectively. As for the essential oil applied on adults, LC(LD)₅₀ was estimated as 9.3 μL mL^− 1 (spray test), 1.9 μL L^− 1 (fumigation test) and 0.6 μg cm^− 2 (tarsal test), and for trans-Anethole as 8.1 μL mL^− 1 (spray test), 2.1 μL L^− 1 (fumigation test) and 0.4 μg cm^− 2 (tarsal test). The time needed to achieve 50% mortality after application of LC(LD)₉₉ of the essential oil was significantly different; for example, in larvicidal assays it ranged from 15 to 235 min depending on the larval instar, and from 9 to 180 min when applied to adults, depending on the mode of application. It was also found that temperature had an important effect on the larvicidal efficacy of the essential oil, and oviposition deterrent activity was studied.The essential oil and trans-Anethole were toxic for Daphnia magna (62–92% mortality) and significantly reduced its fertility at high concentrations (35–50 μL mL^− 1) and long exposure (48 h). However, no negative effect on Daphnia mortality or fertility was found at shorter exposure times (6 h) and/or lower concentrations (20 μL mL^− 1).Based on the results of this study, we can recommend the essential oil from P. anisum as a suitable active substance for potential botanical insecticides.     

Density and distribution of seeds in bottom sediments in Zostera marina beds in Ago Bay,central Japan

The density of Zostera marina L. seeds in bottom sediments was examined to study the reproductive patterns of the Z. marina population in Ago Bay, Mie Prefecture, central Japan.Seeds and seed coats were numerous in Tategami, where the annual type of Z. marina grows. In contrast, seeds were scarce in Hamajima, where the perennial type of Z. marina grows. Bottom sediment was sampled with sediment cores at Tategami in November 2004 and March 2005 to examine density and depth distribution of the seeds. Seeds were found as deep as 8 cm, but no deeper. On the other hand, empty seed coats were found as deep as 16 cm in both months. In the upper layers of the sediment to a depth of 8 cm, the average number of seed coats was 7960 ± 2997 m⁻² in November and 16,318 ± 2922 m⁻² in March. Deeper than 8 cm, the number of seed coats gradually decreased owing to decomposition, and none was found below 16 cm. We used the density of reproductive shoots and number of seeds per spadix in Tategami to estimate the fate of seeds and seed coats of the annual type of Z. marina in bottom sediments: out of the 6000 seeds m⁻² produced annually, 72% disappears from the stand and 28% is buried locally. The density and distribution of Z. marina seeds are among the most important factors in maintenance and propagation of the annual population at Tategami.     

Survivability of indigenous microflora and a Salmonella typhimurium marker strain in poultry mash treated with buffered propionic acid

Buffered propionic acid (BPA) was evaluated as a potential treatment for the elimination of Salmonella spp. in poultry mash. A primary poultry isolate marker strain of Salmonella typhimurium was added as either a broth or in a dry chalk carrier form to poultry mash containing soybean meal as a protein supplement. The mash was supplemented with buffered propionic acid at 2, 4, 6, 8, 10, 20, 30, 50 and 100 g kg⁻¹ diet and samples were enumerated for indigenous aerobic bacteria, fungi and the S. typhimurium marker strain. Total indigenous aerobic bacteria and fungal populations were generally decreased by addition of more than 20 g BPA kg⁻¹, but an addition of 100 g BPA kg⁻¹ mash was usually required to achieve reductions of approximately 90% of indigenous aerobic bacteria and 99% of indigenous fungi. After 7 days of storage, 8 g BPA kg⁻¹ mash also reduced S. typhimurium populations by more than 90% in mash inoculated via chalk, while at least 50 g BPA kg⁻¹ mash was required to provide the same level of reduction in mash inoculated with a liquid culture of S. typhimurium. Although BPA does not appear to be an overly effective antimicrobial agent with respect to indigenous aerobic bacterial populations in animal feed, higher concentrations may have the potential for reducing fungal and Salmonella spp. contamination in poultry mash.     

The effects of arsenic and induced-phytoextraction methods on photosynthesis in Pteris species with different arsenic-accumulating abilities

Arsenic (As) accumulation and photosynthesis occur simultaneously in plants under As exposure. We investigated the effects of As and induced-phytoextraction methods on photosynthesis in two As hyperaccumulators (Pteris vittata and Pteris cretica var. nervosa) and two non-hyperaccumulators (Pteris semipinnata and Pteris ensiformis) under soil culture conditions. Chlorophyll fluorescence parameters (the maximum [F_v/F_m] and actual quantum efficiency [F_PSII]) and the activities of three photosynthetic enzymes (ATPase, ribulose-1, 5-bisphosphate carboxylase [RuBPC] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were measured. Arsenic accumulation and photosynthetic behaviours in response to enhanced-phytoextraction methods (trans-1, 2-cyclobexylenedinitrilotetraacetic acid [CDTA] and phosphorous [P] addition and soil pH adjustment) of P. cretica and P. semipinnata were monitored and compared under conditions of 100 mg As kg⁻¹. Significant decreases in the F_v/F_m (19.9%) and F_PSII (36.1%) were observed in P. vittata when exposed to 100 mg As kg⁻¹ in comparison to the control (0 mg As kg⁻¹). Compared to the control (0 mg As kg⁻¹), the activities of GAPDH increased by 0.5% in P. cretica var. nervosa and decreased by only 8.3% in P. vittata even when both of them were treated with 200 mg As kg⁻¹, whereas a significant decrease, 56.1% and 51.7%, of this enzyme was observed in P. semipinnata and P. ensiformis, respectively, when exposed to 50 mg As kg⁻¹. Compared to the control (0 mg CDTA kg⁻¹ or 0 mg P kg⁻¹), the activities of ATPase increased by 53.7% and 82.7% in P. cretica when exposed to 0.5 g CDTA kg⁻¹ and 50 mg P kg⁻¹, respectively, and an increase of up to 175% was also observed in P. semipinnata when exposed to 600 mg P kg⁻¹. The activities of GAPDH increased by 68.9% and 90.7% in P. cretica when exposed to 2 g CDTA kg⁻¹ and 600 mg P kg⁻¹, respectively, but a decrease of up to 60% was observed in P. semipinnata when exposed to 2 g CDTA kg⁻¹. The uptake of As in P. semipinnata increased by 80.9% and 73.3% when 1 g CDTA kg⁻¹ and 600 mg P kg⁻¹ were added, respectively, compared to the control (0 g CDTA kg⁻¹ or 0 mg P kg⁻¹). It was concluded that GAPDH played an important role in the photosynthesis of As hyperaccumulators under As treatments.     

Oxygen enhances the recovery of Potamogeton maackianus from prolonged exposure to very low irradiance

Recovery ability in relation to carbohydrate content of Potamogeton maackianus growing in two dissolved oxygen concentrations (8 and 2 mg L⁻¹) was investigated during 28 days exposure to very low irradiance (about 0.06 μmol m⁻² s⁻¹). Plant weight remained relatively constant (0.19 g dry wt plant⁻¹) within the initial 21 days in the high oxygen treatment, but decreased to 0.14 g dry wt plant⁻¹ at the end of the experiment. In low oxygen environments, plant weight was similar within the initial 14 days, but decreased to 0.08 g dry wt plant⁻¹ at 21 day. During the experimental period, both soluble sugar and starch contents in shoots decreased with time. Compared to high oxygen treatment, plants in the low oxygen treatment depleted starch more quickly (25 versus 18 mg g⁻¹ at 28 day) but remained a relatively high soluble sugar content (0.9 versus 1.8 mg g⁻¹ at 28 day). After recovery in high light and high dissolved oxygen conditions for 1 week, plant growth rate, new branch number, stem elongation rate and leaf recruitment number were significantly higher in high oxygen than in the low oxygen treatments. These data suggest that the ability of the plant to recover from prolonged exposure to very low irradiance is related to the depletion level of carbohydrate stored in plant tissues, which is regulated by oxygen availability in the water.     

Effect of different dietary energy level intakes on efficiency of estrus synchronization and fertility in Mashona goat does

The objective of the study was to determine the effects of three dietary energy levels: 0.27 (low level: LL); 0.53 (medium level: ML), and 1.06 (high level: HL) MJ ME kg⁻¹ W^0.75 on estrus synchronization and fertility in Mashona goat does. Forty-five multiparous Mashona goat does of average bodyweight 19.9±2.5 kg were randomly allocated in equal numbers to the three dietary energy levels. The diets were made from a complete feed ration providing 9.83 MJ ME kg⁻¹ DM and 15.5% CP kg⁻¹ DM. Does were fed initially during a 60-day pre-synchronization period, and blood samples were collected twice a week for the determination of plasma progesterone concentrations to ascertain ovarian activity. Intramuscular injections of cloprostenol (100 μg each) were administered 11 days apart. Immediately after the second injection of cloprostenol, three fertile bucks were introduced to the does and were left with the does for 21 days. The does were maintained on their dietary treatments throughout gestation except for those does in the LL treatment. Pregnancy was diagnosed 90 days post-mating using an ultrasound scanner. After pregnancy diagnosis, does on the LL treatment were randomly allocated to ML (n=7) and HL (n=8) treatments. During the pre-synchronization period, does on the LL treatment lost 12.3% whereas those on ML and HL treatments gained 2.1 and 28.8% of their initial bodymasses, respectively. The proportion of does exhibiting overt estrus within 96 h after the last cloprostenol injection was significantly lower (P<0.05) for does on the LL treatment (60%) than for those on ML (93%) or HL (100%) treatments, respectively. However, based on plasma progesterone concentrations, the percentage of does on the LL treatment that exhibited ovarian cycles was numerically lower than that of does that were bred (40 versus 73%). Conception, fecundity and twinning rates were significantly lower (P<0.05) on the LL treatment than on the ML and HL treatments. These results indicate that feeding Mashona goat does 0.27 MJ ME kg⁻¹ W^0.75 compared to 0.53 and 1.06 MJ ME kg⁻¹ W^0.75 reduces the expression of estrus, conception, fecundity and twinning rates, and that feeding 0.53 MJ ME kg⁻¹ W^0.75 suffices for optimum reproduction. In addition, the results suggest that cloprostenol administration may induce ovarian cycles in reproductively quiescent does on dietary energy restriction.