Inhibition of ROS-Scavenging Enzyme System Is a Key Event in Tomato Genetic Resistance against Root-Knot Nematodes.

Genetic resistance in plants against incompatible pests is expressed by the activation of an immune system; however, the molecular mechanisms of pest recognition and expression of immunity, although long the object of investigation, are far from being fully understood. The immune response triggered by the infection of soil-borne parasites, such as root-knot nematodes (RKNs), to incompatible resistant tomato plants was studied and compared to the compatible response that occurred when RKNs attacked susceptible plants. In compatible interactions, the invading nematode juveniles were allowed to fully develop and reproduce, whilst that was impeded in incompatible interactions. In crude root extracts, a first assay of reactive oxygen species (ROS)-scavenging enzymatic activity was carried out at the earliest stages of tomato-RKN incompatible interaction. Membrane-bound and soluble CAT, which is the most active enzyme in hydrogen peroxide (H2O2) scavenging, was found to be specifically inhibited in roots of inoculated resistant plants until 5 days after inoculation, with respect to uninoculated plants. The expression of genes encoding for antioxidant enzymes, such as CAT and glutathione peroxidase (GPX), was not always inhibited in roots of nematode-infected resistant tomato. Therefore, the biochemical mechanisms of CAT inhibition were further investigated. Two CAT isozymes were characterized by size exclusion HPLC as a tetrameric form with a molecular weight of 220,000 dalton and its subunits (55,000 dalton). Fractions containing such isozymes were tested by their sensitivity to both salicylic acid (SA) and H2O2. It was evidenced that elevated concentrations of both chemicals led to a partial inactivation of CAT. Elevated concentrations of H2O2 in incompatible interactions have been suggested to be produced by membrane-bound superoxide anion generating, SOD, and isoperoxidase-enhanced activities. Such partial inactivation of CAT has been depicted as one of the earliest key metabolic events, which is specifically associated with tomato immunity to RKNs. Enhanced ROS production and the inhibition of ROS-scavenging systems have been considered to trigger all the metabolic events leading to cell death and tissue necrosis developed around the head of the invading juveniles by which this special type of plant resistance is exerted.

Resistance to Plant Parasites in Tomato Is Induced by Soil Enrichment with Specific Bacterial and Fungal Rhizosphere Microbiome.

Commercial formulations of beneficial microbes have been used to enrich the rhizosphere microbiome of tomato plants grown in pots located in a glasshouse. These plants have been subjected to attacks by soil-borne parasites, such as root-knot nematodes (RKNs), and herbivores, such as the miner insect Tuta absoluta. The development of both parasites and the symptoms of their parasitism were restricted in these plants with respect to plants left untreated. A mixture, named in the text as Myco, containing plant growth-promoting rhizobacteria (PGPR), opportunistic biocontrol fungi (BCF), and arbuscular mycorrhizal fungi (AMF) was more effective in limiting pest damage than a formulation containing the sole AMF (Ozor). Therefore, Myco-treated plants inoculated with RKNs were taken as a model for further studies. The PGPR contained in Myco were not able to reduce nematode infection; rather, they worsened symptoms in plants compared with those observed in untreated plants. Therefore, it was argued that both BCF and AMF were the microorganisms that colonized roots and stimulated the plant immune system against RKNs. Beneficial fungi colonized the roots by lowering the activities of the defense supporting enzymes endochitinases and ß-1,3-glucanase. However, as early as three days after nematode inoculation, these enzyme activities and the expression of the encoding pathogenesis-related genes (PR-2, PR-3) were found to be enhanced in roots with respect to non-inoculated plants, thus indicating that plants had been primed against RKNs. The addition of paclobutrazol, which reduces salicylic acid (SA) levels in cells, and diphenyliodonium chloride, which inhibits superoxide generation, completely abolished the repressive effect of Myco on nematode infection. Inhibitors of copper enzymes and the alternative cyanide-resistant respiration did not significantly alter resistance induction by Myco. When Myco-treated plants were subjected to moderate water stress and inoculated with nematodes, they retained numbers of developed individuals in the roots similar to those present in regularly watered plants, in contrast to what occurred in roots of untreated stressed plants that hosted very few individuals because of poor nutrient availability.

Tomato Root Colonization by Exogenously Inoculated Arbuscular Mycorrhizal Fungi Induces Resistance against Root-Knot Nematodes in a Dose-Dependent Manner.

Arbuscular mycorrhizal fungi (AMF) are generally recognized to induce plant growth and prime plants against soil-borne parasites, such as plant parasitic nematodes. However, the effectiveness of commercial formulates containing AMF has been questioned. Increasing amounts per plant of one commercial AMF-containing formulate, reported in the text as Myco, were used to detect the effects on growth of tomato plants and the resistance induced against root-knot nematodes (RKNs) The doses used per plant (0.5, 1.0, 2.0 g, reported as Myco1, Myco2, Myco3, respectively) were soil-drenched to growing potted plants; the effects of such treatments were analyzed both in plants not inoculated or inoculated by Meloidogyne incognita juveniles. Consistent increases in plant weight were apparent as soon as 7 days only after Myco2 treatments. Moreover, only treatments with Myco2 induced a consistent repression of the nematode infection observed in untreated plants. Conversely, treatments with Myco1 and Myco3 did not produce such an early growth improvement; some plant weight increase was observable only at 28 dpt. Accordingly, such Myco doses did not restrict the level of infestation observed in untreated plants. Control of infection was dependent on the dose of Myco provided to plants five days before nematode inoculation. About one month after all Myco treatments, several areas of roots were found to be colonized by AMF, although in Myco2-treated plants, three genes involved in the AMF colonization process (SlCCaMK, SlLYK9, and SlLYK13) were found to be over-expressed already at 7 dpt; over-expression was generally less consistent at 14 and 21 dpt. The expressions of two key genes of plant defense, the hypersensitive cell death inducer PR4b gene and the glutathione peroxidase-encoding GPX gene, were monitored in roots of Myco2-treated plants 3 and 7 days after nematode inoculation. PR4b was over-expressed and GPX was silenced in treated plants with respect to untreated plants. The repressive effect of Myco2 treatment against RKN infection was completely abolished when Myco2 suspensions were autoclaved to sterilization or treated with the potent anti-fungal agent amphotericin B, thus indicating that the biological control agents contained in the commercial formulate were living fungi.

Exploring the link between star and planet formation with Ariel.

The goal of the Ariel space mission is to observe a large and diversified population of transiting planets around a range of host star types to collect information on their atmospheric composition. The planetary bulk and atmospheric compositions bear the marks of the way the planets formed: Ariel's observations will therefore provide an unprecedented wealth of data to advance our understanding of planet formation in our Galaxy. A number of environmental and evolutionary factors, however, can affect the final atmospheric composition. Here we provide a concise overview of which factors and effects of the star and planet formation processes can shape the atmospheric compositions that will be observed by Ariel, and highlight how Ariel's characteristics make this mission optimally suited to address this very complex problem.

Bringing high spatial resolution to the far-infrared: A giant leap for astrophysics.

The far-infrared (FIR) regime is one of the wavelength ranges where no astronomical data with sub-arcsecond spatial resolution exist. None of the medium-term satellite projects like SPICA, Millimetron, or the Origins Space Telescope will resolve this malady. For many research areas, however, information at high spatial and spectral resolution in the FIR, taken from atomic fine-structure lines, from highly excited carbon monoxide (CO), light hydrides, and especially from water lines would open the door for transformative science. A main theme will be to trace the role of water in proto-planetary discs, to observationally advance our understanding of the planet formation process and, intimately related to that, the pathways to habitable planets and the emergence of life. Furthermore, key observations will zoom into the physics and chemistry of the star-formation process in our own Galaxy, as well as in external galaxies. The FIR provides unique tools to investigate in particular the energetics of heating, cooling, and shocks. The velocity-resolved data in these tracers will reveal the detailed dynamics engrained in these processes in a spatially resolved fashion, and will deliver the perfect synergy with ground-based molecular line data for the colder dense gas.

Epigenetic and Metabolic Changes in Root-Knot Nematode-Plant Interactions.

Two wild-type field populations of root-knot nematodes (Mi-Vfield, Mj-TunC2field), and two isolates selected for virulence in laboratory on resistant tomato cultivars (SM2V, SM11C2), were used to induce a resistance reaction in tomato to the soil-borne parasites. Epigenetic and metabolic mechanisms of resistance were detected and compared with those occurring in partially or fully successful infections. The activated epigenetic mechanisms in plant resistance, as opposed to those activated in infected plants, were detected by analyzing the methylated status of total DNA, by ELISA methods, and the expression level of key genes involved in the methylation pathway, by qRT-PCR. DNA hypo-methylation and down-regulation of two methyl-transferase genes (CMT2, DRM5), characterized the only true resistant reaction obtained by inoculating the Mi-1.2-carrying resistant tomato cv Rossol with the avirulent field population Mi-Vfield. On the contrary, in the roots into which nematodes were allowed to develop and reproduce, total DNA was generally found to be hyper-methylated and methyl-transferase genes up-loaded. DNA hypo-methylation was considered to be the upstream mechanism that triggers the general gene over-expression observed in plant resistance. Gene silencing induced by nematodes may be obtained through DNA hyper-methylation and methyl-transferase gene activation. Plant resistance is also characterized by an inhibition of the anti-oxidant enzyme system and activation of the defense enzyme chitinase, as opposed to the activation of such a system and inhibition of the defense enzyme glucanase in roots infested by nematodes.

Induction of SA-signaling pathway and ethylene biosynthesis in Trichoderma harzianum-treated tomato plants after infection of the root-knot nematode Meloidogyne incognita.

KEY MESSAGE: Salicylic acid-signaling pathway and ethylene biosynthesis were induced in tomato treated with Trichoderma harzianum when infected by root-knot nematodes and limited the infection by activation of SAR and ethylene production. Soil pre-treatment with Trichoderma harzianum (Th) strains ITEM 908 (T908) and T908-5 decreased susceptibility of tomato to Meloidogyne incognita, as assessed by restriction in nematode reproduction and development. The effect of T. harzianum treatments on plant defense was detected by monitoring the expression of the genes PR-1/PR-5 and JERF3/ACO, markers of the SA- and JA/ET-dependent signaling pathways, respectively. The compatible nematode-plant interaction in absence of fungi caused a marked suppression of PR-1, PR-5, and ACO gene expressions, either locally or systemically, whilst expression of JERF3 gene resulted unaffected. Conversely, when plants were pre-treated with Th-strains, over-expression of PR-1, PR-5, and ACO genes was observed in roots 5 days after nematode inoculation. JERF3 gene expression did not change in Th-colonized plants challenged with nematodes. In the absence of nematodes, Trichoderma-root interaction was characterized by the inhibition of both SA-dependent signaling pathway and ET biosynthesis, and, in the case of PR-1 and ACO genes, this inhibition was systemic. JERF3 gene expression was systemically restricted only at the very early stages of plant-fungi interaction. Data presented indicate that Th-colonization primed roots for Systemic Acquired Resistance (SAR) against root-knot nematodes and reacted to nematode infection more efficiently than untreated plants. Such a response probably involves also activation of ET production, through an augmented transcription of the ACO gene, which encodes for the enzyme catalyzing the last step of ET biosynthesis. JA signaling and Induced Systemic Resistance (ISR) do not seem to be involved in the biocontrol action of the tested Th-strains against RKNs.

Use of Chemicals and Biological Control Agents as Activators of Plant Defense Against Endoparasitic Sedentary Nematodes.

Full compatible interactions between crop plants and endoparasitic sedentary nematodes (ESNs) lead to severe infestation of the roots and plant growth impairing, as well as to the increase of nematode population in the soil that is a threat for the next planting crop. In the absence of activators, basic plant defense is overcome by nematode secretion of effectors that suppress defense gene expression, inhibit ROS generation and the oxidative burst used by plants to hamper nematode feeding site settlement and limit its development and reproduction. Activators can be exogenously added as a preventive measure to prime plants and strengthen their defense against ESNs. Activators can be an array of antioxidant compounds or biocontrol agents, such as mutualist microorganisms living in the rhizosphere (biocontrol fungi (BCF), arbuscular mycorrhizal fungi (AMF), plant growth-promoting bacteria (PGPB), etc.). In this chapter, methods are described for usage of both salicylic acid (SA) and its methylated form (Met-SA), and BCF/AMF as elicitors of resistance of vegetable crops against root-knot nematodes (RKNs). The rhizosphere-living BCF/AMF were recovered from commercial formulates pre-incubated in suitable growth media and provided exclusively as soil drench of potted plants. The plant hormones SA and Met-SA were provided to plants as soil drench, foliar spray, and root dip. It is indicated that activators' dosages and plant age are crucial factors in determining the success of a pre-treatment to reduce nematode infection. Therefore, dosages should be expressed as amounts of activators per g of plant weight at treatment. Thresholds exist above which dosages start to work; overdoses were found to be toxic to plants and useless as activators.

Natural genetic and induced plant resistance, as a control strategy to plant-parasitic nematodes alternative to pesticides.

Plant-parasitic nematodes are pests of a wide range of economically important crops, causing severe losses to agriculture. Natural genetic resistance of plants is expected to be a valid solution of the many problems nematodes cause all over the world. Progress in resistance applications is particularly important for the less-developed countries of tropical and subtropical regions, since use of resistant cultivars may be the only possible and economically feasible control strategy in those farming systems. Resistance is being considered of particular importance also in modern high-input production systems of developed countries, as the customary reliance on chemical nematicides has been restricted or has come to an end. This review briefly describes the genetic bases of resistance to nematodes in plants and focuses on the chances and problems of its exploitation as a key element in an integrated management program. Much space is dedicated to the major problem of resistance durability, in that the intensive use of resistant cultivars is likely to increasingly induce the selection of virulent populations able to "break" the resistance. Protocols of pest-host suitability are described, as bioassays are being used to evaluate local nematode populations in their potential to be selected on resistant germplasm and endanger resistant crops. The recent progress in using robust and durable resistances against nematodes as an efficient method for growers in vegetable cropping systems is reported, as well as the possible use of chemicals that do not show any unfavorable impact on environment, to induce in plants resistance against plant-parasitic nematodes.

Bio-control agents activate plant immune response and prime susceptible tomato against root-knot nematodes.

Beneficial microorganisms are generally known to activate plant defense against biotic challenges. However, the molecular mechanisms by which activated plants react more rapidly and actively to pests remain still largely unclear. Tomato plants pre-treated with a mixture of beneficial bio-control agents (BCAs), as soil-drenches, were less sensitive to infection of the root-knot nematode (RKN) Meloidogyne incognita. To unravel the molecular mechanisms of this induced resistance against RKNs, we used qRT-PCR to monitor the expression, in tomato roots and leaves, of 6 key defense genes. Gene transcripts were detected until the 12th day after BCA treatment(3, 7, 8, 12 dpt) and3 and 7 days after nematode inoculation of pre-treated plants. Early after BCA treatment, the salicylic acid (SA)-dependent pathogenesis related gene (PR-gene), PR-1b, marker of the systemic acquired resistance (SAR), was systemically over-expressed. Another PR-gene, PR-5, was over-expressed at later stages of BCA-plant interaction, and only in roots. Activation of defense against RKNs was attested by the early up-regulation of 4 genes (PR-1, PR-3, PR-5, ACO) in pre-treated plants after inoculation. Conversely, the expression of the JA/ET-dependent gene JERF3 did not increase after nematode inoculation in primed plants. A catalase gene (CAT)was highly over-expressed by nematode infection, however, this over-expression was annulled at the earliest stages or limited at the later stages of infection toBCA-treated roots. Enzyme activities, such as glucanase and endochitinase, were enhanced in roots of pre-treated inoculated plants with respect to plants left not inoculated as a control. These findings indicate that BCA interaction with roots primes plants against RKNs. BCA-mediated immunity seems to rely on SA-mediated SAR and to be associated with both the activation of chitinase and glucanase enzyme activities and the inhibition of the plant antioxidant enzyme system. Immunity is triggered at the penetration and movements inside the roots of the invading nematode juveniles but probably acts at the feeding site building stage of nematode infection.

Systemic acquired resistance activation in solanaceous crops as a management strategy against root-knot nematodes.

BACKGROUND: Activators of systemic acquired resistance (SAR), such as salicylic acid (SA) and its synthetic functional analogues benzo(1,2,3)thiadiazole-7-carbothionic acid-S-methyl ester (BTH) and 2,6-dichloroisonicotinic acid (INA), were tested on tomato, eggplant and pepper for the control of the root-knot nematode Meloidogyne incognita. Effects on plant fitness, nematode reproduction and root galling were screened in relation to different methods of application, to different applied dosages of chemicals and to different plant growth stages. Dosages applied to plants were in relation to plant weights. These chemicals were also tested for their possible nematotoxic activity in vitro. RESULTS: Soil drenches of SA and INA and root dip application of SA and BTH inhibited nematode reproduction, at specific dosage ranges, without affecting plant growth. SA and INA were able to reduce root galling as well. Foliar sprays of both SA and BTH were ineffective against nematode attacks. Plants tolerated SA more than the other chemicals tested. BTH at elevated concentrations increased the mortality of nematode juveniles and reduced egg hatching in vitro. CONCLUSIONS: SAR activators at concentrations suitable for different plant growth stages and applied by the proper method can possibly be included in IPM programmes for nematode management.

Expression of tomato salicylic acid (SA)-responsive pathogenesis-related genes in Mi-1-mediated and SA-induced resistance to root-knot nematodes.

The expression pattern of pathogenesis-related genes PR-1, PR-2 and PR-5, considered as markers for salicylic acid (SA)-dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre-treated with SA and subsequently infected with root-knot nematodes (RKNs) (Meloidogyne incognita). PR-1 was up-regulated in both roots and shoots of SA-treated plants, whereas the expression of PR-5 was enhanced only in roots. The over-expression of PR-1 in the whole plant occurred as soon as 1 day after SA treatment. Up-regulation of the PR-1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA-treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR-1, PR-2 and PR-5 was also examined in the roots and shoots of susceptible and Mi-1-carrying resistant tomato plants infected by RKNs. Nematode infection produced a down-regulation of PR genes in both roots and shoots of SA-treated and untreated plants, and in roots of Mi-carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR-1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR-like response to RKNs in tomato.

Coexistence of two different mutations in codon 12 of the Kras gene in colorectal cancer: Report of a case supporting the concept of tumoral heterogeneity.

Evaluation of the mutational status of KRAS is a crucial step for the correct therapeutic approach in treating advanced colorectal cancer as the identification of wild-type KRAS tumors leads to more specific and less toxic treatments for patients. Although several studies have highlighted the differences between primary and metastatic tumors, the possibility of two or more mutations in the same codon has seldom been reported. The present study reports an additional case of an advanced adenocarcinoma of the colon showing two somatic mutations (p.G12D and p.G12V) in the same codon (codon 12) of exon 2 of the KRAS gene, thus supporting the possibility of two differing clonal origins of the tumor. Although the clinical significance of multiple mutations remains unknown at present, based on the limited data available in the literature, this rare event appears to be associated with a more aggressive disease, as in the present case. This case report demonstrates the existence of intratumoral heterogeneity and the coexistence of distinct clones within a tumor that may have profound clinical implications for disease progression and therapeutic responses.

Autotransfusäo sanguinea em artroplastia total do quadril / Autologous blood transfusion in total hip arthroplasty

Foram feitas coletas de sangue no período pré-operatório em 91 pacientes submetidos a 107 artroplastias totais do quadril, com o objetivo de ser utilizado para autotransfusao. Foram coletadas 220 unidades de sangue e reinfundidas 197 (média de 2,05 e 1,84 unidades por operaçäo, respectivamente). Houve necessidade de complementaçäo com sangue homólogo em quatro ocasioes (3,62 por cento). Os critérios de indicaçäo de transfusäo foram baseados na evoluçäo clínica dos pacientes. Apenas o valor do volume globular baixo näo foi considerado como indicaçäo de transfusäo. Näo houve complicaçöes que pudessem ser atribuídas à coleta ou utilizaçäo do sangue autólogo. A evoluçäo clínica dos pacientes foi normal em todos os casos. Näo houve caso de infecçäo. Notou-se um esforço coletivo por parte de todos da equipe cirúrgica para reduçäo de transfusäo sanguínea. Isso resultou em menor utilizaçäo do estoque do banco de sangue e maior segurança para os pacientes, evitando-se os riscos da transfusäo de sangue homólogo.