Replant problems in South Tyrol: role of fungal pathogens and microbial population in conventional and organic apple orchards

The South Tyrol, the main apple-growing area in Italy, is characterised by intensive soil cultivation. Previous investigations have revealed the existence of replant disorders although the main causes of these have not been evaluated. A survey was carried out in this area with two main aims: to evaluate the role of soil-borne pathogens in apple replant disease and to compare the impact of organic and conventional management on replant diseases caused by soil-borne pathogens. The experimental sites were chosen to obtain three pairs of contiguous conventional and organic apple orchards. There was a high level of organic matter in both the organic and the conventional apple orchards with no appreciable differences in humic fractions. The soil sickness test with apple seedlings revealed a significant reduction in growth for all soil samples as compared to the peat control. The growth score on soil samples from organic orchards was significantly higher than that obtained on conventional soil. Total fungal population in soil samples was positively correlated to the apple seedlings growth score and negatively correlated to the frequency of root colonisation. Most of the root-colonising pathogens belonged to the well-known root rot complex, Rhizoctonia solani and Pythium spp. being the most pathogenic on apple seedlings.     

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to “quasi-natural” products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.     

Oviposition and tarsal chemoreceptors of the cabbage root fly are stimulated by glucosinolates and host plant extracts

The role of glucosinolates in the oviposition behaviour of the cabbage root fly,Delia radicum (L.) (Diptera, Anthomyiidae) was investigated using egg counts and electrophysiological recordings from tarsal contact chemoreceptors. The glucosinolates present both inside and on the surface of cauliflower leaves were determined. The total amounts obtained with the two methods differed by a factor of 100. The extract of the leaf surface contained about 60 μg per g leaf extracted (gle), the total leaf extract 7.5 mg per gle. The glucosinolate patterns of the two extracts were qualitatively similar, but the ratios of the content of individual glucosinolates showed considerable differences. The D sensilla on segment 3 and 4 of the tarsus ofD. radicum females were shown to contain a sensitive receptor cell for glucosinolates. In contrast, the receptor cells of the D sensilla of the other segments did not respond in a dose dependent way to these compounds. The glucosinolate receptors were found to be especially sensitive to glucobrassicin, gluconasturtiin and glucobrassicanapin with thresholds of about 10⁻⁸ M to 10⁻⁹ M. Large differences (up to two orders of magnitude) were observed among the different glucosinolates. A significant correlation was found between the behavioural discrimination index and the electrophysiological results. But no obvious correlation existed between the chemical nature of the glucosinolate side chain (e.g. indole, aromatic and aliphatic groups), and their stimulatory activity. However, a significant correlation was found between the overall length of the side chain and the biological activity. Although the flies discriminated clearly between model leaves with and without glucosinolates, a clear dose response curve was only obtained for the indole glucosinolate glucobrassicin. Since the most stimulatory fraction of the surface extract contained no glucosinolates, it was concluded that other compounds, in addition to glucosinolates, do play an important role for the stimulation of oviposition.     

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns

Introduction

We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains.

Materials and methods

We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37 °C for 150 min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA).

Results

IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p < 0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p < 0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p < 0.05) and 50% (p < 0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p < 0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p < 0.05).

Conclusion

Susceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.     

Subtidal Underwater Rock Communities of the White Sea: Structure and Interaction with Bottom Flow

The taxonomic composition and quantitative characteristics of typical subtidal rock communities of the White Sea (biocenosis of Laminaria and biocenosis of Lithothamnion and the sponges Polymastia arctica) were studied. The hydrodynamic activity of the near-bottom water layer within and beyond the communities was estimated using the plaster structures technique. A comparative analysis of our results and the literature data revealed three main types of interactions between the benthic community and the water flow: turbulation of the water flow (mussel banks), smoothing of turbulent pulsations (Laminaria community), and an insignificant effect on the water flow (community of Lithothamnion and sponges).     

Induction of a Crassulacean acid like metabolism in the C4 succulent plant,Portulaca oleracea L.: physiological and morphological changes are accompanied by specific modifications in phosphoenolpyruvate carboxylase

The induction of a Crassulacean acid like metabolism (CAM) was evidenced after 21–23 days of drought stress in the C₄ succulent plant Portulaca oleracea L. by changes in the CO₂ exchange pattern, in malic acid content and in titratable acidity during the day–night cycle. Light microscopy studies also revealed differences in the leaf structure after the drought treatment. Following the induction of the CAM-like metabolism, the regulatory properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the enzyme responsible for the diurnal fixation of CO₂ in C₄ plants but nocturnal in CAM plants, were studied. The enzyme from stressed plants showed different kinetic properties with respect to controls, notably its lack of cooperativity, higher sensitivity to L-malate inhibition, higher PEP affinity and lower enzyme content on a protein basis. In both conditions, PEPC's subunit mass was 110 kDa, although changes in the isoelectric point and electrophoretic mobility of the native enzyme were observed. In vivo phosphorylation and native isoelectrofocusing studies indicated variations in the phosphorylation status of the enzyme of samples collected during the night and day, which was clearly different for the control and stressed groups of plants. The results presented suggest that PEPC activity and regulation are modified upon drought stress treatment in a way that allows P. oleracea to perform a CAM-like metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Field tests of cis-regulatory variation at the prairie vole avpr1a locus: association with V1aR abundance but not sexual or social fidelity

The neuropeptide vasopressin and its receptor V1aR are broadly implicated in social behavior and play a central role in several key aspects of male mating tactics in voles. In the prairie vole, a microsatellite in the cis-regulatory region of the gene encoding V1aR (avpr1a) provides a potential genetic basis for individual variation in neural phenotype and behavior; recent studies found that allele length predicts V1aR expression and male social attachment in the laboratory. Here, we explore the relationship between avpr1a microsatellite length, V1aR neural phenotype, and field measures of monogamy and fitness in male prairie voles. We found significant effects of allele length on V1aR expression in structures integral to pairbond formation. These effects did not, however, translate to differences in mating tactics or reproductive success. Together, these data suggest that, while length polymorphism in the avpr1a microsatellite influences neuronal phenotype, this variation does not contribute significantly to male reproductive success and field behavior. We propose that previously reported behavioral effects may be mediated primarily by sequence variation at this locus, for which allele length is an imperfect proxy. By combining genetic, neuronal and ecological approaches, these data provide novel insights into the contribution of genotype to natural diversity in brain and behavior.     

Ectomycorrhizal syntheses with Picea abies and three fungal species: a case study on the use of an in vitro technique to identify naturally occurring ectomycorrhizae

Ectomycorrhizal syntheses between Picea abies and the fungal associates Scleroderma citrinum, Boletus luridus, and Tricholoma vaccinum were carried out using Melin's Erlenmeyer flask technique. The symbioses of S. citrinum were characterized by a mantle composed of an outer prosenchymatous and an inner synenchymatous layer. The mantles of B. luridus and T. vaccinum were solely prosenchymatous. Rhizomorphs were produced in all treatments, but only in association with S. citrinum were they differentiated with additional, enlarged hyphae. All synthesized ectomycorrhizae were white or whitish to light orange and greyishorange. On large-scale root sampling in two differing Picea abies forests in Switzerland, nine out of a total of 22 morphological types of ectomycorrhizae were white or yellow in colour and were, therefore, comparable with the synthesized ectomycorrhizae. These nine natural types generally had distinct mantle features (irregular synenchyma, gelatinous matrix, cystides, thick-walled hyphae), but mostly lacked clamp connections. Synthesized ectomycorrhizae, on the other hand, lacked distinct mantle characteristics and always had clamp connections. Natural and synthesized white or yellow ectomycorrhizae did not coincide morphologically and thus identification of the fungal partners of natural symbioses by means of in vitro-synthesis with potential ectomycorrhizal fungi was not possible in the present study.     

Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: Functional divergence of the dilysine ER retrieval motif in plant cells

Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N- and C-termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (–KK–COOH rather than the prototypic –KKXX–COOH or –KXKXX–COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (––X–X–K/R/D/E––; where are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C-termini of other GPAT8s from various plant species, further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.     

Photosynthesis and phage: early studies on phosphorus metabolism in photosynthetic microorganisms with 32P,and how they led to the serendipic discovery of 32P-decay suicide of bacteriophage

During my PhD thesis research (1946–1949), I explored the effects of light on the uptake of ³²P-labeled inorganic phosphate (P_i) by cells of photosynthetic bacteria and microalgae, and the dynamics of P turnover between low and high molecular weight cell constituents. The results were interpreted as evidence for the conversion of light energy to the chemical energy of phosphorylated compounds. The experimental results also suggested to me that the precursors of the P in DNA bacteriophages of Escherichia coli must be low molecular weight phosphorylated compounds present within the host cells and led to the design of an experiment to determine the conservation of ³²P of an infecting phage particle in its numerous progeny. The experiment envisaged was never conducted because phage labeled with ³²P of high specific activity showed unexpected loss of viability. Thus, by serendipity, ‘suicide’ of phage due to ³²P-β decay was discovered. ³²P-decay ‘suicide’ provided a technique that was useful for analysis of phage genetic structure and replication. This memoir describes the unusual circumstances leading to the decisive role of serendipity in revealing an extraordinary phenomenon. This revised version was published online in June 2006 with corrections to the Cover Date.     

Leishmania donovani: dyskinetoplastid cells survive and proliferate in the presence of pyruvate and uridine but do not undergo apoptosis after treatment with camptothecin

We have shown that treatment with luteolin in leishmanial cells causes loss of mt-DNA and induces apoptosis through mitochondria dependent pathway [Sen, N., Das, B.B., Ganguly, A., Banerjee, B., Sen, T., Majumder, H.K., 2006. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells. Experimental Parasitology, in press]. Here, we report that mitochondrial DNA depleted leishmanial cells require exogenous sources of pyruvate and uridine to survive and proliferate. The presence of pyruvate and uridine in a growing media help them to produce sufficient amount of glycolytic ATP to maintain the mitochondrial membrane potential in the absence of their functional ETC. Treatment of wild type cells with CPT causes generation of ROS that leads to apoptosis. But unlike the normal cells ROS was not generated in these mt-DNA depleted cells after treatment with CPT. Taken together we have shown for the first time that dyskinetoplastid cells are auxotrophic for pyruvate and uridine and apoptosis cannot be induced in these cells in the presence of CPT. Therefore, the presence of mitochondrial DNA is absolutely necessary for the cytotoxicity of CPT in kinetoplastid parasites.