Allyl isothiocyanate: an alternative chemical expellant for sampling earthworms

Allyl isothiocyanate (AITC), a natural breakdown product of glucosinolates in many Cruciferae and a component imparting the sharp taste to mustard, was tested for its effectiveness as a chemical expellant for sampling earthworms. Testing was performed in an arable field with earthworm populations dominated by Lumbricus terrestris Linnaeus, 1758 and Aporrectodea tuberculata (Eisen, 1874). The optimal concentration was found by comparing concentrations ranging from 5 to 250 mg l⁻¹ in water. Total biomass and numbers of earthworms collected increased hyperbolically with increasing AITC concentration, with the highest biomass and numbers collected using 100 mg l⁻¹ AITC. Biomass and numbers of earthworms collected using 250 mg l⁻¹ AITC, but not 150 or 200 mg l⁻¹ AITC, were significantly less than with 100 mg l⁻¹ AITC. Less earthworm biomass was collected by hand sorting than with chemical expulsion using 100 mg l⁻¹ AITC, but the number of earthworms collected by the two methods were not different. A comparison of hand sorting and 100 mg l⁻¹ AITC expulsion using analysis of similarities (ANOSIM) showed that the two methods produced samples differing in the distribution of both numbers and biomass of species-by-size classes. Hand sorting collected more of the smallest size class of L. terrestris and the largest size classes of A. tuberculata than AITC expulsion, whereas AITC expulsion recovered more of the largest size classes of L. terrestris than hand sorting. When 100 mg l⁻¹ AITC expulsion was compared with chemical expulsion using 200 mg l⁻¹ formalin, no differences were found in the total number, total biomass or in the species-by-size class distribution of the earthworms collected. This suggests that the AITC method may be a favorable alternative to formalin expulsion for sampling earthworms. Further studies under other environmental conditions and with other species of earthworms are warranted to establish its general utility.     

Halophyte root powder: an alternative biofertilizer and carrier for saline land

ABSTRACT

The present investigation was based on the hypothesis that the endophytes residing in the roots of halophytes have better adaptation to saline conditions. Six halophytic herbs were collected from Khewra salt range (EC = 4.7 dS m^?1 and SAR = 25.7). From these herbs, root pieces of Cenchrus ciliaris were shade dried; finely ground to powder and three plant growth promoting rhizobacteria (PGPR), Bacillus cereus, Pseudomonad moraviensis, and Stenotrophomonas maltophilia, were isolated. Root powder in sterilized and unsterilized forms was added in the saline-sodic field on wheat and mixed with soil in pot experiment with induced NaCl (150 mM). Sterilized root powder increased organic matter NO₃-N and P contents of soil and leaves, fresh weight, sugar content, and yield attributes. The root powder application in unsterilized form significantly decreased EC, SAR, and Na content of field soil with concomitant increase in soil and leaves K, P, and NO₃-N. The farmer’s benefit was increased by 33% at yield. Root powder-induced salt tolerance was mediated by the PGPR (residing inside the root) through increased growth and better physiological adaptations. It is inferred that root powder harboring the PGPR may be an alternative to biofertilizer with longer shelf life and may also serve as carrier for the preparation of effective biofertilizer for saline land using other PGPR bio-inoculants.     

Scheme for validation of an analytical protocol: semiautomated Kjeldahl nitrogen determination

Definitive validation of an analytical protocol is not always possible. A scheme for performing an alternative type of validation is presented. The scheme is tested on a commonly used chemical procedure for assaying total nitrogen. This alternative validation scheme uses 5 approaches: tests of robustness to recovery of working standards, assay of fortified in-house control materials to assess recovery from typical material, prescribed means of measuring within-and between-batch precision of the assay of typical material, comparison with alternative assay procedures, and determination of analogous certified reference materials to enable calculation of total error. The scheme uses the results of the 5 approaches to obtain a performance profile for the assay. The scheme does not rely on involved statistical modeling or data treatment, yet provides the laboratory worker with a systematic approach to a within-laboratory validation of an analytical protocol.     

Mineral oil barrier is an effective alternative for suppression of damage by white snails

Purpose: Citrus white snail, Helicella candeharica Pfeiffer (Panpulmonata: Helicidae) is one of the most important orchard pests. In this study, the effectiveness of mineral oil was compared with molluscicide baits such as metaldehyde, ferricole (iron phosphate) and a snail-repellent paint in a commercial citrus orchard in northern Iran to reduce access of citrus white snails to citrus trees.

Materials and methods: The number of snails on citrus trees was monitored and counted 10 days after the application of the treatments, and at an interval of 6–8 days up to harvest time.

Results: In the first study, the mineral oil and repellent paint treatments reduced a number of snails best. In the second study, using metaldehyde and mineral oil barrier, again the mineral oil barrier reduced snails best. The cost of each treatment during one season per hectare was calculated at 55, 153, 124 and 120?$/ha for mineral oil, iron phosphate, snail-repellent paint and metaldehyde, respectively.

Conclusions: Mineral oil is an effective alternative for chemical compounds for reducing access by H. candeharica to citrus trees.     

Measuring the rhizodeposition of carbon by rice: an approach based on carbon flux observations

ABSTRACT

Rhizodeposition is an important component of carbon cycling in terrestrial ecosystems. However, there remains tremendous uncertainty in its quantification due to the methodological limitations. In the present study, we propose a method to evaluate the rhizodeposition by plants by observing carbon flux. We investigated the ecosystem CO₂ flux variability and calculated the rhizodeposition of carbon by the rice rhizosphere, by using the carbon flux, meteorological data, and biomass observation from 2003 to 2011 at the Taoyuan Agro-ecological Experimental Station, a representative subtropical paddy ecosystem. Our data indicated that the process of rhizodeposition is the major reason for the discrepancy between the biomass and net primary productivity of the paddy ecosystem under intensive human interference. Both the amount and ratio of rhizodeposition of carbon in this paddy ecosystem were assessed; this provides important theoretical and methodological support for further investigating rhizodeposition by rice under field conditions. The rhizodeposition amount in the growing season of early rice, late rice, and for the entire planting period was 0.52–2.56, 0.74–3.75, and 1.61–5.24 t ha^?1, respectively, with the corresponding mean (±SD) rhizodeposition ratios of 23.16 ± 8.87%, 28.16 ± 12.94%, and 27.00 ± 9.3%. This method enabled us to calculate rhizodeposition under in situ conditions, and the results showed that the growing season of late rice was the primary period for rhizodeposition in rice ecosystem.     

Structural equation modelling: an alternative for assessing causal relationships in threatened plant populations

Structural equation modelling (SEM) is a powerful tool to explore and contrast hypotheses on causal relationships among variables using observational data. It constitutes an alternative to experimental approaches that is especially useful in the conservation of small populations where the implementation of treatments may have a negative effect on population viability. We are presently applying SEM to study the factors that condition reproductive success, seed emergence and plantlet survival in several plant species. We are also using model comparisons through multi-sample analysis to assess the implications of different microhabitats on the viability of a population. The most outstanding advantages of this tool are the global perspective used in the study of complex problems, the ability to discern the essential from the accessory, and the possibility of evaluating one's own hypotheses. The basic procedure, the limitations of this method and further applications in conservation and management are also discussed.     

Development of an enzyme-linked immunosorbent assay for the veratrum plant teratogens: cyclopamine and jervine

Veratrum californicum was responsible for large losses of sheep grazing high mountain ranges in central Idaho in the 1950s. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey-faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids cyclopamine (1) and jervine (2) were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine (1) and jervine (2) are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation-stage embryonic development, producing cyclopia and holoprosencephaly. Although losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep and other species. However, the benefits to biomedical research using cyclopamine (1) as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) to detect and measure cyclopamine (1) and jervine (2) was developed using polyclonal antibodies produced in ewes. The limits of detection of the assay were 90.0 and 22.7 pg for cyclopamine (1) and jervine (2), respectively. This assay was used for the detection and measurement of cyclopamine (1) spiked into sheep blood. The simple extraction-ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine (1) and jervine (2) and will be beneficial to pharmacological studies and livestock diagnostics.     

Optimising reintroduction success by delayed dispersal: Is the release protocol important for hare-wallabies?

The transfer of threatened animals from one location to another in order to benefit the species is a technique frequently used by animal conservation managers. However, very few of these relocations have experimentally assessed the relative merits and disadvantages of commonly used release techniques. The premise examined in this study was that a higher degree of site fidelity should be advantageous for an individual through a soft release protocol. Two species of hare-wallaby, mala (Lagorchestes hirsutus) and merrnine (Lagostrophus fasciatus), were fitted with radio-collars and reintroduced in August 2001 onto Peron Peninsula in Western Australia. These threatened species were reintroduced using two release strategies (soft versus hard release), and their movements and body condition were monitored, for a period of four weeks post-release, using radio-telemetry and trapping. Each species averaged decreases in body condition index when exposed to both experimental release protocols. A total of 11 animals (32%) emigrated from the release sites, with 10 of these animals being male. Importantly, no relationship was found between site fidelity and release method, although body condition was significantly higher in mala which dispersed, and there was observational evidence for a male-biased dispersal for merrnine. Although the number of released individuals of each species was low, this study demonstrates an interesting trend which indicates that soft release techniques do not necessarily to confer an advantage to the successful immediate establishment and survival of either hare-wallaby species in the short term. We recommend that managers involved with species reintroduction programs consider the costs and potential outcomes of designing and installing soft release enclosures.     

Development and validation of an optical SPR biosensor assay for tylosin residues in honey

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.     

Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.     

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.     

Use of digestate as an alternative to mineral fertilizer: effects on growth and crop quality

Nowadays, the recycling of organic wastes represents a key factor for improving soil and crop quality with socio-economic and environmental benefits. In recent years, the production of digestate significantly increased with a crescent need to be sustainable disposal. The use of digestate as soil amendment has been widely studied but few researches evaluated the digestate effects on crop quality. Since it has been proven that digestate increased soil fertility, we hypothesized that it could increase crop quality as organic fertilizers do. To verify this hypothesis, we analyzed the effects of two digestates on Cucumber quality. Cucumber was chosen because it is an important old crop used worldwide as fresh food, and in cosmetic and pharmaceutic industries. Results showed that digestates increased its content of phenols and flavonoids with antioxidant activity. Neohesperidine and hesperitin with well-known antioxidant, anti-inflammatory and anti-carcinogenic properties as well as naringin and narirutin with the power of reducing the risk of atherosclerosis and cardiovascular complications were manifested only in digestate amended cucumbers. These results highlighted the potentiality of digestates not only as crop growth promoter but also as phytochemical inducers representing a good option for the production of high-quality food with reduced chemical mineral fertilizer inputs.     

Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines

The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm(-)(1)) and basic pH values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.     

Enzymatic assay for the determination of olive oil polyphenol content: assay conditions and validation of the method

A new spectrophotometric assay for the determination of the polyphenolic content of olive oil is presented. It is a substrate-recycling assay for phenolic compounds that employs tyrosinase in the presence of excess NADH. The reaction of various phenols with the enzyme produces an o-quinone, which is detected by recycling between reactions with the enzyme and NADH. The method offers some advantages over the classical methods employed to determine the polyphenolic content of olive oil, that is, ease and reproducibility of the analysis, highly increased sensitivity, and selectivity toward phenolic compounds. The amount of total polyphenols was determined in virgin olive oils both with the Folin-Ciocalteu reagent and with the proposed enzymatic method. The results suggest a better estimation of the polyphenol content, as compared with the colorimetric method. This has to be attributed to the different reactivities of the two methods toward phenols and catechols. Finally, the enzymatic method demonstrates that there is a linear relationship between the olive oil phenolic content and the antioxidative capacity of oil extracts.     

Development and fast screening of salbutamol residues in swine serum by an enzyme-linked immunosorbent assay in Taiwan

The analysis of salbutamol in swine serum is the more practical basis for large scale surveillance programs in Taiwan. Objectives of the study were to develop a new assay and to compare with a commercially available kit in field test screens. A simple and reliable enzyme-linked immunosorbent assay (ELISA) to monitor the presence of beta-agonist, salbutamol, in 1,358 field samples of swine serum that were collected from local meat markets was described. The method proved to be suitable and sensitive for the detection of beta-agonist residues caused by growth promoting dosage. The limit of detection of the developed ELISA directly performed on diluted serum was 0.25 ng/mL. The application and the results of two ELISA kits (homemade and commercially available) for the screening of salbutamol were presented. For further confirmation, all samples that showed to be ELISA positive for salbutamol residues were analyzed by GC-MS. Adopting 1 ng/mL salbutamol as a cutoff value, the commercial beta-agonist ELISA had a sensitivity of 89.2% and a specificity of 86.7% versus GC-MS at a cutoff of 1 ng/mL. The homemade salbutamol ELISA had a sensitivity of 81.1% and a specificity of 98.6% and gave a low proportion of false-positive rate results. The reliability of the developed kit in terms of the percentage of false-positive rate results is evaluated. In conclusion, a sensitive, specific salbutamol ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of beta-agonists.     

Screening for antioxidant activity in edible plant products: comparison of low-density lipoprotein oxidation assay, DPPH radical scavenging assay, and Folin-Ciocalteu assay

Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis. Antioxidants that prevent LDL from oxidizing may reduce atherosclerosis. This study investigated LDL antioxidant activity in edible plant products for development of dietary supplementation to prevent atherosclerosis. Fifty-two kinds of edible plants were extracted using 70% aqueous ethanol solution, and the antioxidant activity of the extracts, which inhibit human LDL oxidation induced by copper ion, was determined on the basis of the oxidation lag time and represented as epigallocatechin 3-gallate equivalent. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content were also measured for comparisons with antioxidant activity in LDL. Plant products showing the greatest activity in LDL oxidation assay were akamegashiwa (Mallotus japonicus) leaf, Japanese privet (Ligustrum japonicum) leaf, green tea [Camellia sinensis (L.) O. Kuntze], and astringent persimmon (Diospyros kaki). The present study revealed high levels of LDL antioxidant activity in plant products for which such activity levels are underestimated in the DPPH radical scavenging assay and Folin-Ciocalteu assay.     

Biochar derived from dairy cattle carcasses as an alternative source of phosphorus and amendment for soil acidity

Two pot experiments were conducted to evaluate biochar derived from dead dairy cattle as a mineral fertilizer, especially phosphorus (P) fertilizer, and to clarify the effect of particle size of biochar on plant growth (Zea mays L.) and P uptake. To produce the biochar, body parts of dead cattle were placed in a charring chamber and allowed to char at 450°C for 4 h. The biochar was of high pH and rich in major plant nutrients, especially P. Application of fine biochar (< 1 mm) increased P uptake by the corn plants grown in soil of low available P status. As a result, plant growth was improved following biochar application and dry matter production was also increased. The effect of the biochar application on the P uptake and plant growth was promoted by the application of mineral nitrogen (N) fertilizer. Soil analysis after harvest indicated that the biochar application increased soil pH, available P and exchangeable calcium (Ca) and magnesium (Mg) compared with the soil before seeding, while soil available N and exchangeable potassium (K) were considerably decreased. The decrease in the soil available N was incomprehensible, because the result of the mass balance given by the difference between input as the applied N from the biochar and fertilizer N and output as the N uptake by the plants was positive. We observed a similar result in the mass balance of K to the case of N. The medium (2–4 mm) and coarse (> 4 mm) grade biochar did not significantly affect plant growth, because P uptake was not, or was only slightly, increased by the application of these biochars. Dissolution of P from the coarser biochars was probably slower than that of the fine biochar. The lower dissolution of P from the medium and coarse biochars was supported by the lower P absorption efficiency of these biochars compared with that of the fine biochar and superphosphate. The effect of fine biochar on plant growth and P uptake was similar to that of superphosphate. We can therefore conclude that fine biochar derived from cattle carcasses is an effective source of P fertilizer and amendment for soil acidity. The N and K contents in the biochar, although relatively high, cannot be relied upon as a mineral fertilizer. Further studies are needed to assess whether the N and K contents of the biochar indicate it can be regarded as a useful fertilizer.     

Development of a compound-specific ELISA for flufenoxuron and an improved class-specific assay for benzoylphenylurea insect growth regulators.

This study describes immunochemical approaches for the compound-specific detection of flufenoxuron and class-specific detection of benzoylphenylurea (BPU) insecticides. With the aim of developing a highly specific immunoassay for flufenoxuron, a hapten was synthesized by introducing a spacer arm at the 2,6-difluoro substituent aromatic ring of a flufenoxuron derivative. An IC(50) value of 2.4 ppb was obtained for flufenoxuron, with detection of the other four BPUs being more than 4000-fold less sensitive. For the development of class-specific ELISA for five BPUs, a new approach was used for the hapten preparation in which a butanoic acid linkage was introduced into the 3,5-dichloro-substituted aniline ring of chlorfluazuron analogue. Although the resultant ELISA still exhibited slightly differing cross-reactions for these five BPUs, this method had broader specificity than the previously reported polyclonal antibody-based ELISA. Spike and recovery studies for five BPUs in soil and water indicated that both the compound- and class-specific ELISAs were able to quantitatively detect BPU residues in soil and water. This study also provided additional insights into the influence of the immunizing hapten structure on the specificities of the antibodies obtained.     

Identification and quantification of Fusarium oxysporum in planta and soil by means of an improved specific and quantitative PCR assay

The proper identification and quantification of F. oxysporum populations inhabiting soil and plant rhizosphere niches are of importance for soil microbial ecology and plant pathology. In this study, we report the improvement of a PCR protocol for the specific identification of the F. oxysporum species complex and its conversion into a real-time qPCR assay for the quantification up to 1 pg of the fungus DNA in soil and different plant tissues. The amplification efficiency, sensibility and reproducibility of qPCR assays were not influenced by presence of non-target DNA from either plant or soil. The applicability of the newly developed qPCR protocol for F. oxysporum population studies was demonstrated using the technique for quantifying the fungus in different complex environmental samples. The use of the qPCR protocol allowed to accurately quantify up to 25 pg of F. oxysporum/g of naturally infested field soil, as well as to identify significant differences in the amount of F. oxysporum DNA in roots of different chickpea cultivars grown in a field soil infested with diverse pathogenic and nonpathogenic F. oxysporum populations. This qPCR protocol may be especially important for studies on soil microbial ecology and plant pathology since it provides a new opportunity for analyzing F. oxysporum populations and their interactions with the soil microflora, environment and plant host genotypes.     

Development and evaluation of an immunological approach for the identification of novel acetyl coenzyme-A carboxylase inhibitors: assay optimization and pilot screen results

Cyclohexanediones, aryloxyphenoxypropionates, indolizidinediones, and triazinediones are four known structural classes of herbicides that inhibit acetyl coenzyme-A carboxylase (ACCase; EC 6.4.1.2). An immunological study to determine the potential of ACCase inhibitor-specific monoclonal antibodies as screening tools to identify novel lead chemistry was undertaken. Using two cyclohexanedione-specific monoclonal antibodies (mAb A and mAb B; Webb, S. R.; Hall, J. C. J. Agric. Food Chem. 2000, 48, 1210-1218) and three different cyclohexanedione hapten coating conjugates, competitive indirect enzyme-linked immunosorbent assays (ciELISA) were developed. Cross-reactivity of the monoclonal antibodies with four structural classes of ACCase inhibitors revealed that the ciELISA using mAb A and a modified cyclohexanedione hapten coating conjugate detected analogues from all four known classes of ACCase inhibitors. A pilot screen using this ciELISA format identified two novel ACCase inhibitors, demonstrating the potential for antibodies as rapid and cost-effective screening tools for identifying novel lead chemistry in pesticide discovery programs.