Detection and quantification of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in irrigation waters

A quantitative RT real-time PCR method was developed for the detection and quantification of Tomato mosaic virus (ToMV) in irrigation waters. These have rarely been monitored for the presence of plant pathogenic viruses, mostly due to the lack of efficient and sensitive detection methods. The newly developed method presented here offers a novel approach in monitoring the health status of environmental waters. ToMV was reliably detected at as low as 12 viral particles per real-time PCR reaction, which corresponds to the initial concentration of approximately 4.2 × 10^?10 mg (6,300 viral particles) of ToMV per ml of sample. The sensitivity of the method was further improved by including the Convective Interaction Media^® (CIM) monolithic chromatographic columns for quick and efficient concentration of original water samples. Seven out of nine water sources from different locations in Slovenia tested positive for ToMV, after concentrating the sample. Four samples tested ToMV-positive without the concentrating procedure. The presence and integrity of infective ToMV particles in the original sample, as well as in the chromatographic fraction, was confirmed using different methods from test plants, DAS ELISA to electron microscopy and real-time PCR. In this study, we propose a unique and simple diagnostic scheme for rapid, efficient, and sensitive monitoring of irrigation waters that could also be adopted for other plant, human or animal viruses.     

Comparison of Various Phosphate Stabilisation Agents for the Immobilisation of Ni and Zn in Sewage Sludge

This paper reports the results of the treatment of polychlorinatedbiphenyl (PCB) contaminated sandy soils (100 mg kg^-1 Aroclor 1242) with the Fenton advanced oxidation process (AOP). The results obtained in the various assays permitted the optimization of conditions as follows: 5% H₂O₂; 100 ppm of Fe³⁺; and a ratio of sandy soil mass/volume of oxidizing solution (m/V) of 1/3 g mL^-1. In addition, these tests established the need for agitation and dispensed with the need for heat. The results obtained confirm that the oxidation process occurs in solid phase (on the PCBs adsorbed to soil particles), producing 98% elimination of the original PCB structure and 82% dechlorination, all within a reaction time of 72 hr. The degree of elimination was found to be dependent on the level of congener chlorination and the process displays a pseudo first order kinetics. In addition, the Fenton chemical oxidation process may be complemented by subsequent aerobic biological degradation which, after 15 days, produces 72% mineralization of the products generated during the chemical oxidation process.     

Molecular diversity of ‘Candidatus Phytoplasma pyri’ isolates in Slovenia

A European quarantine organism ‘Candidatus Phytoplasma pyri’ causing devastating pear decline disease has been reported to affect pear trees in several European countries. In this study a multilocus sequence analysis was successfully used to gain detailed insight into the molecular diversity of thirty closely related ‘Candidatus Phytoplasma pyri’ isolates from different orchards in Slovenia. Among three genomic regions analyzed, the 16S/23S rRNA intergenic spacer region was the most conserved among Slovenian isolates with 99.7 % sequence identity, yielding only three distinct genotypes. On the other hand, five different genotypes were detected when analyzing secY and aceF genomic regions that shared sequence identity of 94.8 and 97.2 %, respectively. Six of the detected genotypes, specifically four in the secY region and one in each of the two other analyzed genomic regions, were unique for Slovenia. At least eight different haplotypes were found with multilocus sequence analysis, indicating high molecular diversity among Slovenian ‘Ca. P. pyri’ isolates. Haplotypes were clustered into two major clusters, separated by at least 45 mutations. No connection was established between haplotype occurrence and cultivar type.     

Driving forces for agroforestry uptake in Mediterranean Europe: application of the analytic network process

The factors that determine the implementation of four alternative agroforestry practices or no agroforestry on a theoretical 200 ha farm in Mediterranean Europe were examined using an analytic network process (ANP) model. The four agroforestry practices considered were implementation of a form of (i) high natural and cultural value agroforestry, (ii) agroforestry with high value trees, and agroforestry for (iii) arable and (iv) livestock systems. The ANP model was developed in a participatory manner through a systematic series of quantitative questionnaires and workshops with agroforestry researchers. In general, all the Mediterranean agroforestry systems were associated with high benefits and opportunities, but also with high costs and high risks. The greatest benefits were attributed to high natural and cultural value agroforestry systems, which greatly contributed to the highest priority of this system. Overall ranking of priorities for the agroforestry management alternatives show robustness in the sensitivity analysis. The “no agroforestry” land use became the preferred option when costs were given a weighting of 0.50 or greater.     

Testing the influence of sediment granulometry on heterotrophic respiration with a new laboratory flow-through system

Purpose

Increased sedimentation due to land use intensification is increasingly affecting carbon processing in streams and rivers around the globe. This study describes the design of a laboratory-scale flow-through incubation system as a tool for the rapid estimation of sediment respiration. The measurements were compared with those obtained using an in situ closed chamber respiration method. The influence of sediment size on respiration rates was also investigated.

Materials and methods

Measurements were conducted on a pre-alpine gravel-bed river sediment separated into the following grain size fractions: > 60 mm (14.3%), 60–5 mm (60.2%), 5–2 mm (13.7%), 2–0.063 mm (11.1%) and <0.063 mm (0.6%). Concurrently, in situ and laboratory measurements were carried out on a naturally heterogeneous sediment. In situ respiration was determined in closed chambers as O₂ consumption over time, while in the laboratory, respiration was determined using flow-through respiration chambers. Oxygen concentrations were measured using a fibre-optic oxygen meter positioned at the inflow and outflow from the chamber.

Results and discussion

The mean respiration rates within naturally mixed riverbed sediments were 1.27 ± 0.3 mg O₂ dm^?3 h^?1 (n = 4) and 0.77 ± 0.1 mg O₂ dm^?3 h^?1 (n = 3) for the flow-through chamber system and closed chamber system, respectively. Respiration rates were statistically significantly higher in the flow-through chamber system (t test, p < 0.05), indicating that closed chamber measurements underestimated the oxygen consumption within riverbed sediments. Sediment grain size was found to significantly affect respiration rates in both systems (ANOVA, p < 0.001) with the fine sediment fraction (particle size <0.063 mm) having the highest respiration rate (r_flow-through = 51 ± 23 mg O₂ dm^?3 h^?1). The smallest fractions (2–0.063 and <0.063 mm), which represent approximately 12% of total sediment volume, contributed 60% of total respiration.

Conclusions

The study demonstrated that flow-through respiration chambers more accurately estimate the respiration rate within riverbed sediments than in situ closed chambers, since the former experiment imitates the natural conditions where continuous interstitial flow occurs in the sediments. We also demonstrated that fine sediments (<5 mm) substantially contribute to heterotrophic respiration in the studied gravel-bed river.

     

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.     

Comparison of methods for the estimation of best parent heterosis among lines developed from interspecific sunflower germplasm

Pre-breeding and elite breeding are two steps in creating high yielding sunflower hybrids that differ in well established procedures and selection methods. However, a methodology that bridge efficient use of introgression lines as product of pre-breeding procedures and their crossing to elite inbreed lines, is not yet very well established. Therefore, the development of cost- and time-efficient methods for the determination of best parent heterosis and the use of best inbred lines in crosses with introgression lines for obtaining high-yielding and stable hybrids is highly desirable. In this regard, sixteen Cytoplasmic Male Sterile (CMS) inbred lines (A) derived from four heterogeneous interspecific lines originating from three annual: H. debilis silvestris (DEB-SIL), H. praecox runyoni (PRA-RUN), H. deserticola (DES) and one perennial H. resinosus (RES) wild species were evaluated. Seven agronomic traits were measured over a period of 2 years and 38 DNA loci were analysed, in order to compare four different methods for the estimation of best parent heterosis (BPH). New inbred lines were characterized by Principal Component Analysis (PCA) of morphological traits and Principal Coordinate Analysis (PCoA) of molecular marker data. Line?×?tester mating design was used to evaluate General Combining Ability (GCA), while Genetic Distance (GC) estimated by markers was evaluated as a predictor of BPH by Locally Weighted Sequential Smoothing (LOESS). Analysis of combining ability is one of the most important tools breeders use to identify superior inbred lines on the basis of their performance in hybrid combinations. Results obtained in this research show that PCA of morphological and PCoA of molecular marker data on parental lines are generally in agreement with GCA effects for examined traits. GD versus BPH relationships indicate that intermediate to high GD between parental lines was optimal for best heterotic effects of most traits. In this study, we show that the combination of the PCA of morphological data, PCoA of molecular marker data and GD between parental lines is fast and affordable, giving the most important information for parental choice of introgression and elite lines in sunflower breeding programs.     

Molecular diversity of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma mali’ and ‘<Emphasis Type="Italic">Ca</Emphasis>. P. prunorum’ in orchards in Slovenia

The plant parasitic nematode Longidorus poessneckensis found in Austria, the Czech Republic, Germany, Poland and the Slovak Republic was molecularly characterized. Mitochondrial genes encoding cytochrome c oxidase subunit I (COI) and nicotinamide dehydrogenase subunit 4 (nad4), the D2 and D3 expansion segments of 28S rRNA and Internal transcribed spacer 1 (ITS1) rRNA were sequenced for 16 L. poessneckensis populations. Six haplotypes of COI and five haplotypes of nad4 were determined. Nucleotide intraspecific variation was up to 17.1% for the partial sequenced COI gene and up to 17.7% for the partial sequenced nad4 gene, the latter being the highest up to date known intraspecific variation in this genus. The analyses of multiple amino acid sequence alignments of mitochondrial genes revealed low variability (0–2.4%) for COI gene and high divergence (0–7.6%) for nad4 gene. Intraspecific sequence diversity for the D2-D3 of 28S rRNA gene was up to 1.2% and for ITS1 rRNA gene was up to 1.6%. It has been hypothesized, that during the Last Glacial Maximum, L. poessneckensis populations probably persisted in refuge areas in the Carpathian Mountains and subsequently expanding from these areas and colonizing other European regions.