Detection of Koi herpesvirus: impact of extraction method,primer set and DNA polymerase on the sensitivity of polymerase chain reaction examinations

Since virus isolation is seldom successful, KHV infection is commonly detected by PCR examination. A number of different PCR assays have been described in recent years. However, at present no commonly accepted PCR method is used amongst different laboratories. The aim of this study was to check if the examination of infected fish by different PCR methods yielded comparable results. We used tissue samples of three KHV‐infected koi, one KHV‐infected common carp, one KHV‐infected goldfish and one non‐infected common carp. DNA was extracted with DNAzol Reagent, High Pure PCR Template DNA Preparation Kit and QIAamp DNA Mini Kit. The DNA was tested by PCR with different combinations of published primer sets –KHV‐F and ‐R, KHV‐Gray‐2F and ‐2R and KHV‐TKf and ‐TKr – plus different DNA polymerases – a standard Taq DNA polymerase, a Platinum (hotstart) Taq DNA polymerase and a Platinum (hotstart) Pfx DNA polymerase with proofreading activity. The different extraction methods produced DNA solutions with different yields of DNA and different degrees of homogeneity. Also, the sensitivity of the PCR depended on the choice of the primer set and polymerase. Not all infected fish could be identified with all methods; there were large differences in the sensitivity between methods.     

RFID tags for identifying and verifying agrochemicals in food traceability systems

The objective of this paper is to identify what data should be stored in an automatic recording system to trace the use of agrochemicals. RFID tags are proposed as the most appropriate storage systems. The essential information to store on an RFID tag is as follows: country of registration, chemical type, unique registration number of an agrochemical, container size, specific gravity, unit of measure, and a digital signature. Digital signatures address issues of verification of data integrity and security—a major concern of the agrochemical industry. Detailed data will be drawn from publicly available databases of approved pesticides. Encoding schemes have been designed which can record all of the essential information on commonly available cheap RFID labels. A prototype system to record and transfer data in a traceability system is developed, including hardware and software aspects. The user interface of the system is presented with a sample sequence of user screens to assist the loading of a full pack of agrochemical tagged with an RFID transponder. An experimental trial with practicing agrochemical professionals was undertaken. The user interface proved effective and acceptable. During the trial, more than 250 product identification cycles with RFID were carried out without failure.     

Comparison of two short DNA barcoding loci (COI and COII) and two longer ribosomal DNA genes (SSU & LSU rRNA) for specimen identification among quarantine root-knot nematodes (Meloidogyne spp.) and their close relatives

Root-knot nematodes (Meloidogyne spp.) are important pests of numerous crops worldwide. Some members of this genus have a quarantine status, and accurate species identification is required to prevent further spreading. DNA barcoding is a method for organism identification in non-complex DNA backgrounds based on informative motifs in short DNA stretches (≈600 bp). As part of the EU 7th Framework project QBOL, 15 Meloidogyne species were chosen to compare the resolutions offered by two typical DNA barcoding loci, COI and COII, with the distinguishing signals produced by two ribosomal DNA genes (small and large subunit rDNA; SSU?≈?1,700 and LSU?≈?3,400 bp). None of the four markers distinguished between the tropical species Meloidogyne incognita, M. javanica and M. arenaria. Taking P ID (Liberal) values ≥0.93 as a measure for species delimitation, the four mtDNA and rDNA markers performed well for the tropical Meloidogyne species complex, M. enterolobii, M. hapla, and M. maritima. Within cluster III A (Holterman et al. Phytopathology, 99, 227–235, 2009), SSU rDNA did not offer resolution at species level. Both mtDNA loci COI and COII did, whereas for LSU rDNA a longer fragment (≥700 bp) is required. The high level of mitochondrial heteroplasmy recently reported for M. chitwoodi (Humphreys-Pereira and Elling Nematology, 15, 315–327, 2013) was not found in the populations under investigation, suggesting this could be a regional phenomenon. For identification of RKNs, we suggest the combined use of SSU rDNA with one of three other markers presented here.     

Insecticidal activity of plant-derived extracts against different economically important pest insects

With the aim of selecting potential botanical insecticides, seven plant extracts (Daphne mucronata (Family: Thymelaeaceae), Tagetes minuta (Asteraceae), Calotropis procera (Apocynaceae), Boenninghausenia albiflora (Rutaceae), Eucalyptus sideroxylon (Myrtaceae), Cinnamomum camphora (Lauraceae) and Isodon rugosus (Lamiaceae)) were screened for their toxic effects against four important agricultural pest insects, each representing a separate insect order; pea aphids of Acyrthosiphon pisum (Hemiptera), fruit flies of Drosophila melanogaster (Diptera), red flour beetles of Tribolium castaneum (Coleoptera), and armyworms of Spodoptera exigua (Lepidoptera). Aphids were the most susceptible insect with 100% mortality observed after 24 h for all the plant extracts tested. Further bioassays with lower concentrations of the plant extracts against aphids, revealed the extracts from I. rugosus (LC₅₀ 36 ppm and LC₉₀ 102 ppm) and D. mucronata (LC₅₀ 126 ppm and LC₉₀ 198 ppm) to be the most toxic to aphids. These most active plant extracts were further fractionated into different solvent fractions on polarity basis and their insecticidal activity evaluated. While all the fractions showed considerable mortality in aphids, the most active was the butanol fraction from I. rugosus with an LC₅₀ of 18 ppm and LC₉₀ of 48 ppm. Considering that high mortality was observed in aphids within 24 h of exposure to a very low concentration of the butanol fraction from I. rugosus, we believe this could be exploited and further developed as a potential plant-based insecticide against sucking insect pests, such as aphids.     

Is salt stress of faba bean (Vicia faba) caused by Na+ or Cl– toxicity?

The effect of sodium chloride (NaCl), sodium sulfate (Na₂SO₄), and potassium chloride (KCl) on growth and ion concentrations of faba bean (Vicia faba L. cv. Troy) was studied. After 14 or 15 d of isoosmotic treatment with 100 mM NaCl or 75 mM Na₂SO₄, respectively, plants developed toxicity symptoms. These symptoms were characterized by local and nonchlorotic wilting spots, which later turned to black, necrotic spots. In contrast to NaCl or Na₂SO₄ treatment, plants treated with 100 mM KCl did not show these symptoms. The symptoms occurred on those leaves that accumulated highest concentrations of Na⁺ and showed highest Na⁺ : K⁺ ratios. Our results indicate that Na⁺ toxicity inducing K⁺ deficiency is responsible for the spot necrosis of faba bean. Additionally, chlorotic symptoms occurred. The concentrations of Na⁺ and Cl^– were determined in chlorotic leaves and in isolated chloroplasts. The reduction of chlorophyll in leaves after NaCl exposure may be explained in terms of high Cl^– concentrations in the chloroplasts and appears to depend on high Na⁺ concentrations. Chlorotic toxicity symptoms can be avoided by additional Mg²⁺ application.     

Local response of bacterial densities and enzyme activities to elevated atmospheric CO2 and different N supply in the rhizosphere of Phaseolus vulgaris L.

Altered flux of labile C from plant roots into soil is thought to influence growth and maintenance of microbial communities under elevated atmospheric CO₂ concentrations. We studied the abundance and function of the soil microbial community at two levels of spatial resolution to assess the response of microorganisms in the rhizosphere of the whole root system and of apical root zones of Phaseolus vulgaris L. to elevated CO₂ and high or low N supply.

At the coarser resolution, microbial biomass C, basal respiration and phospholipid fatty acid (PLFA) patterns in the rhizosphere remained unaffected by elevated CO₂, because the C flux from the whole root system into soil did not change [as shown by Haase, S., Neumann, G., Kania, A., Kuzyakov, Y., Römheld, V., Kandeler, E., 2007. Elevation of atmospheric CO₂ and N-nutritional status modify nodulation, nodule carbon supply, and root exudation of Phaseolus vulgaris L. Soil Biology & Biochemistry 39, 2208–2221]. At a higher spatial resolution, more low-molecular-weight compounds were released from apical root zones under elevated CO₂. Thus, at an early stage of plant growth (12 days after sowing), elevated CO₂ induced an increase of enzyme activities (xylosidase, cellobiosidase and leucine-aminopeptidase) in the rhizosphere soil of apical root zones. At later stages of plant growth (21 days after sowing), however, enzyme activities (those above as well as N-acetyl-β-glucosaminidase and phosphatase) decreased under elevated CO₂. The abundance of total and denitrifying bacteria in the rhizosphere soil of apical root zones was unaffected by CO₂ elevation or N supply. Plant age seemed to be the main factor influencing the density of the bacterial community. In conclusion, the soil microbial community in the apical root zone responded to elevated CO₂ by altered enzyme regulation (production and/or activity) and not by greater bacterial abundance.     

Contribution of wheat endogenous and wheat kernel associated microbial endoxylanases to changes in the arabinoxylan population during breadmaking

Wheat kernel associated endoxylanases consist of a majority of microbial endoxylanases and a minority of endogenous endoxylanases. At least part of these enzymes can be expected to end up in wheat flour upon milling. In this study, the contribution of both types of these endoxylanases to changes in the arabinoxylan (AX) population during wheat flour breadmaking was assessed. To this end, wheat flour produced from two wheat varieties with different endoxylanase activity levels, both before and after sodium hypochlorite surface treatment of the wheat kernels, was used in a straight dough breadmaking procedure. Monitoring of the AX population during the breadmaking process showed that changes in AX are to a large extent caused by endogenous endoxylanases, whereas the contribution of microbial endoxylanases to these changes was generally very low. The latter points to a limited contamination of wheat flour with microbial enzymes during milling or to an extensive inactivation of these wheat flour associated microbial endoxylanases by endoxylanase inhibitors, present in wheat flour. When all wheat kernel associated microbial endoxylanases were first washed from the kernels and then added to the bread recipe, they drastically affected the AX population, suggesting that they can have a large impact on whole meal breadmaking.     

Parasites of cultured and wild brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) in Lampung Bay,Indonesia

A total of 210 Epinephelus fuscoguttatus (brown‐marbled grouper) was examined for parasites. During three consecutive seasons (two rainy and one dry season from 2002 to 2004), 35 specimens each taken from floating net cages of the National Sea Farming Development Centre (Balai Budidaya Laut) and from wild catches in Lampung Bay, South Sumatra, Indonesia were studied. Twenty‐five (cultured grouper) and 30 (wild grouper) parasite species/taxa were identified, with an infracommunity ranging from one to nine (cultured) and three to 14 parasite species (wild), demonstrating a species‐rich parasite fauna even in the cultured fish. Protozoans (1 species), microsporeans (1), myxozoans (1), digeneans (8), monogeneans (5), cestodes (3), nematodes (8), acanthocephalans (2) and crustaceans (6) were found. The most abundant parasites were the monogeneans Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus lantauensis for both, cultured and wild grouper during all seasons. For the cultured fish, the prevalence of monoxenous ectoparasites (e.g. P. epinepheli, P. lantauensis, Capsalidae gen. et sp. indet., Benedenia epinepheli) was in most cases higher than that of heteroxenous endoparasites. This contrasts the wild grouper, where heteroxenous parasites such as Allopodocotyle epinepheli and Raphidascaris sp. occurred at a similar prevalence compared with the fairly abundant Pseudorhabdosynochus spp. No seasonality of infestation was observed for both cultured and wild fish. The high levels of infestation of potentially pathogenic monogeneans throughout the year could result in significant parasite outbreaks at the locality studied.     

Environmental determinants of perch (Perca fluviatilis) growth in gravel pit lakes and the relative performance of simple versus complex ecological predictors

Growth of fish is an important contributor to individual fitness as well as fish production. Explaining and predicting growth variation across populations is thus important from fundamental and applied perspectives, which requires knowledge about the ecological factors involved in shaping growth. To that end, we estimated environment-dependent von Bertalanffy growth models for 13 gravel pit lake populations of Eurasian perch (Perca fluviatilis) from north-western Germany. To identify the main drivers of perch growth, we evaluated the performance of 16 different biotic or abiotic lake variables in explaining growth variation among lakes. In addition, we compared growth predictions from the best-performing model incorporating “complex” variables that require intensive sampling effort, with a model using only “simple”, easily measurable lake variables (e.g. shoreline development factor). The derivation of a simple model aimed at future applications in typically data-poor inland fisheries, predicting expected growth potential from easily measurable lake variables. A model combining metabolic biomass of predators, maximum depth and shoreline development factor performed best in predicting perch growth variation across gravel pits. All three parameters in this model were positively related to perch growth. The best-performing simple model consisted only of the shoreline development factor. Length-at-age predictions from both models were largely identical, highlighting the utility of shoreline development factor in approximating growth potential of perch in gravel pits similar to our study lakes. Our results can be used to inform fisheries management and restoration efforts at existing or newly excavated gravel pit lakes.