Monitoring of the copper persistence on plant leaves using pulsed thermography.

Copper-based fungicides are largely used in agriculture in the control of a wide range of plant diseases. Applied on plants, they remain deposited on leaf surfaces and are not absorbed into plant tissues. Because of accumulation problems and their ecotoxicological profiles in the soil, their use needs to be monitored and controlled, also by using modern technologies to better optimize the efficacy rendering minimum the amount of copper per season used. In this work, we test a novel approach based on pulsed thermography to evaluate the persistence of the copper on plant leaves so that the time between two applications should be the minimum needs. We monitored the thermal response observed on different treatments of both grapevine and tobacco plants over a 3-week period. Our experimental results demonstrate that the new methodological approach based on pulsed thermography can be an effective tool to evaluate in real time the presence of copper on differently treated plants allowing a tentative quantification and, therefore, to optimize its use in the agricultural practices, according also to the European Regulation n. 1107/2009.

Potential Use of Cardunculus Biomass on Pleurotus eryngii Production: Heteroglycans Content and Nutritional Properties (Preliminary Results).

The new perspective of using waste biomass to cultivate mushrooms as a source of protein for human nutrition, in line with the circular economy principles, is receiving increasing attention in the scientific community and represents great wealth in terms of environmental sustainability. Pleurotus eryngii is a mushroom also known as cardunculus mushroom due to its ability to grow on this plant. This study explores the potential intrinsic properties of cardunculus (for example, the presence of inulin in the roots) as raw material for the growth of cardunculus mushrooms, and the influence on heteroglycan content and nutrition parameters of the fruiting bodies. Both mycelium and fruiting bodies were used to determine the heteroglycan content in the presence of inulin or cardunculus roots rich in inulin. To produce heteroglycans from P. eryngii in greater quantities and shorter times without having to wait for the formation of the fruiting bodies, the mycelium could be used. The results showed that the presence of cardunculus biomass positively influences the heteroglycan content of P. eryngii. In terms of nutritional parameters, higher contents of polyphenols, flavonoids, anthocyanins, and antioxidant activity were detected in P. eryngii grown on the cardunculus stem and root substrate. In conclusion, recycling cardunculus biomass to generate growth blocks for edible mushrooms is a winning choice due to the opportunity to use this biomass waste, which is gaining more and more attention due to the increase in cultivated areas and the use of fruiting bodies of P. eryngii as a functional food and source of molecules with potential biological activities.

Antibiotic Activity of a Paraphaeosphaeria sporulosa-Produced Diketopiperazine against Salmonella enterica.

A diketopiperazine has been purified from a culture filtrate of the endophytic fungus Paraphaeosphaeria sporulosa, isolated from healthy tissues of strawberry plants in a survey of microbes as sources of anti-bacterial metabolites. Its structure has been determined by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analyses and was found to be identical to cyclo(L-Pro-L-Phe) purified from species of other fungal genera. This secondary metabolite has been selected following bioguided-assay fractionation against two strains of Salmonella enterica, the causal agent of bovine gastroenteritis. The diketopiperazine cyclo(L-Pro-L-Phe), isolated for the first time from Paraphaeosphaeria species, showed minimum inhibitory concentration (MIC) values of 71.3 and 78.6 µg/mL against the two S. enterica strains. This finding may be significant in limiting the use of synthetic antibiotics in animal husbandry and reducing the emergence of bacterial multidrug resistance. Further in vivo experiments of P. sporulosa diketopiperazines are important for the future application of these metabolites.

Productive infection of bovine papillomavirus type 2 in the placenta of pregnant cows affected with urinary bladder tumors.

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness.

Here, we provide genetic and biochemical evidence indicating that the ability of Rhizobium etli bacteria to efficiently catabolize glutamine depends on its ability to produce reduced glutathione (l-γ-glutamyl-l-cysteinylglycine [GSH]). We find that GSH-deficient strains, namely a gshB (GSH synthetase) and a gor (GSH reductase) mutant, can use different amino acids, including histidine, alanine, and asparagine but not glutamine, as sole source of carbon, energy, and nitrogen. Moreover, l-buthionine(S,R)-sulfoximine, a GSH synthesis inhibitor, or diamide that oxidizes GSH, induced the same phenotype in the wild-type strain. Among the steps required for its utilization, glutamine uptake, occurring through the two well-characterized carriers (Aap and Bra systems) but not glutamine degradation or respiration, was largely reduced in GSH-deficient strains. Furthermore, GSH-deficient mutants of R. etli showed a reduced symbiotic efficiency. Exogenous GSH was sufficient to rescue glutamine uptake or degradation ability, as well as the symbiotic effectiveness of GSH mutants. Our results suggest a previously unknown GSH-glutamine metabolic relationship in bacteria.

Sulphadimethoxine inhibits Phaseolus vulgaris root growth and development of N-fixing nodules.

Sulphonamides contamination of cultivated lands occurs through the recurrent spreading of animal wastes from intensive farming. The aim of this study was to test the effect(s) of sulphadimethoxine on the beneficial N-fixing Rhizobium etli-Phaseolus vulgaris symbiosis under laboratory conditions. The consequence of increasing concentrations of sulphadimethoxine on the growth ability of free-living R. etli bacteria, as well as on seed germination, seedling development and growth of common bean plants was examined. We have established that sulphadimethoxine inhibited the growth of both symbiotic partners in a dose-dependent manner. Bacterial invasion occurring in developing root nodules was visualized by fluorescence microscopy generating EGFP-marked R. etli bacteria. Our results proved that the development of symbiotic N-fixing root nodules is hampered by sulphadimethoxine thus identifying sulphonamides as toxic compounds for the Rhizobium-legume symbiosis: a low-input sustainable agricultural practice.

Effects of indole-3-acetic acid on Sinorhizobium meliloti survival and on symbiotic nitrogen fixation and stem dry weight production.

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and alpha-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-beta-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.

The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.

The alae, longitudinal ridges of the lateral cuticle, are the most visible specialization of the Caenorhabditis elegans surface. They are present only in L1 and dauer larvae and in adults. Little is known about the mechanisms through which at the appropriate stages secretion of cuticle components by the seam cells results in the formation of the alae. Here we show that three proteins, each containing a Zona Pellucida domain (ZP), are components of the cuticle necessary for larval alae development: CUT-1 and CUT-5 in dauer larvae and CUT-3 and CUT-5 in L1s. Transcriptional regulation of the corresponding genes contributes to the stage-specific role of these proteins. Larvae with reduced cut-1, cut-3 or cut-5 function not only lack alae but are also larger in diameter due to an increase in the width of the lateral cuticle. We propose a model in which reduction of the body diameter, which occurs in normal L1 and dauer larvae, is the result of a dorso-ventral shrinking of the internal layer of the lateral cuticle and formation of the alae results from the folding of the external layer of the lateral cuticle over the reduced, internal one. Alae of adults appear to form through a different mechanism.

Production of low-lactose milk by means of nonisothermal bioreactors.

The effect of the immobilization time on the activity of immobilized beta-galactosidase from K. lactis was investigated. Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively. Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk. All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes. The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time. The industrial application of these membranes has been discussed in terms of percentage reduction of the production times.

Glutamine utilization by Rhizobium etli.

We undertook the study of the use of glutamine (Gln) as the source of carbon and energy by Rhizobium etli. Tn5-induced mutagenesis allowed us to identify several genes required for Gln utilization, including those coding for two broad-range amino acid transporters and a glutamate dehydrogenase. The isolated mutants were characterized by the analysis of their capacity i) to grow on different media, ii) to transport Gln (uptake assays), and iii) to utilize Gln as the C energy source (CO2 production from Gln). We show that Gln is degraded through the citric acid cycle and that its utilization as the sole C source is related to a change in the bacterial cell shape (from bacillary to coccoid form) and a high susceptibility to a thiol oxidative insult. Both these data and the analysis of ntr-dependent promoters suggested that Gln-grown bacteria are under a condition of C starvation and N sufficiency, and as expected, the addition of glucose counteracted the morphological change and increased both the bacterial growth rate and their resistance to oxidative stress. Finally, a nodulation analysis indicates that the genes involved in Gln transport and degradation are dispensable for the bacterial ability to induce and invade developing nodules, whereas those involved in gluconeogenesis and nucleotide biosynthesis are strictly required.

Auxotrophic mutant strains of Rhizobium etli reveal new nodule development phenotypes.

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix- mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and cytology before considerations of metabolic exchange are made.