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Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is native to South America and has rapidly spread in various countries, becoming one of the major threats to tomato crop (Solanum lycopersicum L.). Its range is constantly expanding, spacing up to the North-Western Himalayan Region of India and in the sub-Saharan Africa area. Although this species has a tremendous potential to develop resistance against many insecticides, its control still relies largely on molecules such as organophosphates, pyrethroids, spinosad and diamides. Sustainable control, either based on the use of predators (i.e mirid bugs) and eggs parasitoids (i.e. Trichogrammatidae) or mating disruption (also combined), has been successful only in specific conditions. In this context, the exploitation of resistance traits from wild relative species could offer another sustainable alternative to synthetic insecticides. It is noteworthy that crop wild relatives harbour an abundant supply of resistance genes for biotic and abiotic stresses. We studied two wild tomato backgrounds, Solanum pennellii (acc. LA0716) and Solanum habrochaites (acc. LA1777), using the corresponding populations of introgression lines (ILs and ILSHs) obtained by crossing the processing tomato inbred varieties M82 and E6203 to the wild accessions LA0716 and LA1777, respectively. These lines are expected to exert defence mechanisms able to hamper the attraction and the larval feeding of T. absoluta. Pest resistance of these wild tomato accessions has been related to naturally occurring allelochemicals present in leaf glandular trichomes such as acyl sugars, 2-tridecanone, or sesquiterpenes, especially zingiberene. We have selected a panel of five lines of both IL populations, and have carried out bioassays to compare their effect on T. absoluta to identify genes putatively involved in resistance to this invasive pest. The number of eggs laid over time unit was used as a measure of adult attraction, while antixenosis/antibiosis was measured by recording survival, development time and pupal weight of feeding larvae on tomato leaves. Our work showed that several ILs exhibit an intermediate resistance level, prompting further studies to characterize the specific gene/s involved. Nonetheless, breeding programmes could benefit from these preliminary results.

Microorganisms play a key role in preserving soil fertility in forest agro-ecosystems. Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers. AM fungi improve plant mineral nutrient acquisition, in exchange for C compounds derived from the photosynthetic process, which can result in a positive host growth response and in an improved response to stress. Previous works, mainly performed in pots, suggest that the effects of the AM symbiosis on plant-water relationships are often subtle, transient, and probably circumstance- and symbiont-specific. Additionally, the mechanisms involved in the mitigation to water stress effects on plant growth by AM fungi are still to be elucidated. Within the framework of a project supported by CNR (AQUA), we studied the impact of the AM symbiosis on tomato (var. San Marzano nano) tolerance to water stress. The first step consisted in developing a protocol for water stress in presence/absence of an AM fungus. Two AM species have been selected to be inoculated separately, i.e. Funneliformis mosseae and Rizophagus intraradices, while non-mycorrhizal plants acted as controls. Leaf water potential (?leaf) and gas exchanges have been monitored until the plants reached a water stress state. By varying the duration of the drought, two levels of water stress were achieved, as confirmed by the measurement of leaf water potential: moderate stress (water potential approximately -0.8 MPa) and severe water stress (leaf water potential below -1.2 MPa), no stress (controls, leaf water potential at -0.3 MPa). Water Use Efficiency (WUE) has been calculated, showing a difference between the two considered AM fungi. At the end of the experiment (when plants have reached a severe stress state), AM colonization degree has been calculated, and morphometric parameters have been considered (e.g., shoot length and diameter, internodal distance, stomatal density). For the following biochemical and molecular analyses, leaves and roots from all the set of plants have been collected. The levels of ABA, proline, H2O2 have been assessed, and the analysis of CAT and SOD activity is being determined. Gene expression analyses (RT-qPCR experiments) will be performed considering genes potentially involved in water stress response (e.g., a tomato gene coding for a dehydrin and LeNCED1) and in mycorrhizal symbiosis (e.g., fungal and plant PT genes), on leaves and root samples respectively. On the basis of the results so far obtained, R. intraradices has been selected for further experiments devoted to verify the impact of the AM symbiosis on the plant responses to biotic stresses (i.e., aphids and nematodes) during drought conditions.

Le piante hanno prodotto nel corso dell’evoluzione svariati meccanismi per difendersi dagli organismi fitofagi. Lo studio delle difese indirette, ossia quelle basate sull’attività dei nemici naturali, va affrontato in modo multidisciplinare, ed ha lo scopo di identificare da un lato le sostanze che regolano l’attrattività verso i nemici naturali, e dall’altro i geni che sono alla base di queste interazioni. Gli afidi inducono in pianta risposte che per tempi e modi si avvicinano alla reazione agli organismi fitopatogeni. Conseguentemente, piuttosto allungati sono i tempi di reazione della pianta rispetto a quelli che si registrano per i fitofagi masticatori. Gli studi di queste interazioni partono sempre da osservazioni sul comportamento degli insetti entomofagi, per individuare da quali componenti dei sistemi multitrofici sono emesse le sostanze volatili in grado di attrarli. Dalla caratterizzazione delle sostanze coinvolte nell’attrattività degli entomofagi, affidata ai chimici delle sostanze naturali e che rappresenta di per se un risultato di carattere applicativo, si cerca di individuare i percorsi metabolici coinvolti nelle risposte della pianta ai fitomizi. Questo passaggio si avvale della collaborazione di fisiologi vegetali che, insieme ai genetisti vegetali, sono in ultima analisi in grado di indicare quei geni che potrebbero essere alla base di queste interazioni e che quindi possono essere indicati per programmi di selezione vegetale. Da studi recentissimi è emerso che la composizione dei composti volatili emessi dalle piante attaccate dagli afidi è solo quantitativamente differente da quella emessa dalle piante sane, sottolineando il minimo danno meccanico che questi insetti provocano ai tessuti vegetali. E’ anche emerso che questi insetti sono in grado di allertare in pianta sia il percorso dell’acido jasmonico che quello dell’acido salicilico. Grazie all’uso di piante geneticamente modificate è stato possibile evidenziare alcuni dei geni coinvolti nella produzione di sostanze volatili attrattive per i nemici naturali degli afidi. Sempre più si richiede una collaborazione interdisciplinare per affrontare questo tipo di studi, al fine di poter arrivare alla definizione di nuove strategie per il controllo ecosostenibile degli insetti dannosi alle piante agrarie.

La T. absoluta è originaria del Sud America dove è considerato il fitofago più importante per la coltura del pomodoro. Nel 2008 si sono avute le prime segnalazioni della sua presenza in Italia. Le prime segnalazioni, quasi tutte contemporanee, ne evidenziavano la presenza solo in alcune regioni e in zone piuttosto limitate (Calabria, Campania, Liguria, Sardegna, Sicilia). La pubblicità derivata da ciò, ha però evidenziato una presenza più diffusa sia all’interno delle regioni già segnalate sia in regioni più o meno limitrofe (Abruzzo, Emilia Romagna, Lazio, Puglia, Toscana). Un esame attento dei campioni raccolti ha inoltre messo in luce, in Liguria, la presenza di un’infestazione contemporanea con un altro fillominatore di nuova introduzione la Keiferia lycopersicella (Walsingham). La T. absoluta è conosciuta con il nome di fillominatrice del pomodoro ma è un insetto capace di alimentarsi e svilupparsi a danno di tutte le solanacee sia quelle coltivate (pomodoro patata, peperone, melanzana) sia quelle selvatiche (Solanum nigrum, Solanum elaeagnifolium, Solanum puberulum, Datura stramonium, Datura ferox etc.). La T. absoluta è un insetto per cui sono raccomandate misure da quarantena, per cui, purtroppo, sono da prevedere risvolti negativi nell’esportazione delle produzioni italiane interessate da questo insetto.

The most common mutation causing Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease, is the hyperexpansion of a polymorphic GAA triplet repeat localized within an Alu sequence (GAA-Alu) in the first intron of the frataxin (X25) gene. GAA-Alu belongs to the AluSx subfamily and contains several polymorphisms in strong linkage disequilibrium either with a subgroup of normal alleles, or with hyperexpanded FRDA-associated alleles. GAA repeat sizes in 300 normal chromosomes (97 from carriers and 203 from controls) were distributed in two separate groups: 83% of them contained between six and 10 triplets (small normal alleles), while the remaining 17% had more than 12 triplets, up to 36 (large normal alleles). Sequence analysis showed that no normal, stable allele contained more than 27 uninterrupted GAA triplets. All longer normal alleles were interrupted by a hexanucleotide repeat (GAGGAA). An allele containing an uninterrupted run of 34 GAA triplets was stably transmitted in four instances, but in one case underwent hyperexpansion to 650 triplets. Overall, our results suggest that the FRDA-associated expanded GAA repeats originate from normal alleles by recurrent expansions of alleles at risk.

In the tritrophic system consisting of tomato, Solanum lycopersicum (L.), the aphid Macrosiphum euphorbiae (Thomas) and its natural enemy, the parasitoid Aphidius ervi (Haliday), it has been shown that the release of volatile organic compounds following aphid attack is responsible for attracting aphid parasitoids in wind tunnel experiments. The main compounds involved in these multitrophic interactions have been characterized and quantified. In this work, the possible activity of such compounds on plant direct defences against the aphid M. euphorbiae was assessed in laboratory tests. The selected compounds were applied to uninfested tomato plants, either by evaporation or contact, and performance of aphids, in terms of plant acceptance, fixing behaviour and aphid development, calculated in standard conditions. The results showed that two compounds, namely methylsalicylate and cishex3en1ol, alter aphid performance. These two compounds have been reported to be those eliciting the best response by A. ervi in terms of flight behavior (wind tunnel bioassay) and antennal stimulation (EAG bioassay).

Bacillus thuringiensis Berliner toxins are highly specific in terms of insect pest targeted. For example, Cry3Bb is active against Coleoptera. As a consequence, in the framework of using genetically modified plants expressing different Bt toxins in sustainable agriculture, there is a growing interest about the impact of these crops on the environment and on non-target organisms. The effects of genetically modified Bt-tomato plants expressing the toxin Cry3Bb on the aphid Macrosiphum euphorbiae (Thomas) and its natural enemies, the generalist predator Macrolophus caliginosus Wagner and the endoparasitoid wasp Aphidius ervi Haliday, were assessed under laboratory conditions. No significant differences between performance of M. euphorbiae were observed on genetically modified tomato plants (line UC82Bt) with respect to their near-isogenic control line (line UC82). Immunoassays did not detect Cry3Bb protein in M. euphorbiae developing on Bt-expressing tomato plants. Similarly, no significant differences were found on the longevity and prey consumption of M. caliginosus when fed aphids reared on UC82Bt or on UC82. Moreover, the genetic modification did not affect the attractiveness of uninfested tomato plants toward A. ervi. It is therefore concluded that Cry3Bb-expressing tomato plants did not show any acute adverse effects on the biological parameters of the non-target herbivore M. euphorbiae or its natural enemies, M. caliginosus and A. ervi, under laboratory conditions. © 2012 Copyright Taylor and Francis Group, LLC.

A greenhouse experiment was setup to study foraging behavior of the bumblebee Bombus terrestris L. on Cry3Bb-expressing genetically modified (GM) eggplants and their near-isogenic control. Commonly, more bumblebees visited GM eggplants compared to near-isogenic control, but this difference was only marginally significant. The mean length of feeding bouts was similar on the two treatments. Neither the number of flowers produced nor their size could explain bumblebees' tendency to prefer GM eggplants. Volatile compounds were extracted from five plants per genotype and separated using gas chromatography. Thirteen compounds were identified and five of them appeared significantly more abundant in GM eggplants. Six of the identified compounds [(+)-limonene, Z-jasmone, p-cymene, α-pinene, methyl-salicilate, and (-)-limonene] were tested in electrophysiological bioassays with antennas detached from young bumblebees, and a response was recorded in all six cases. Experimental results indicate that pollination activity of bumblebees is compatible with this GM eggplant event as a food source and that chemical cues may have an important role in plant identification. The implications for environmental risk assessment of GM plants are discussed. © 2011 Springer Science+Business Media B.V.

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Below ground and above ground plant-insect-microorganism interactions are complex and regulate most of the developmental responses of important crop plants such as tomato. We investigated the influence of root colonization by a nonmycorrhizal plant-growth-promoting fungus on direct and indirect defenses of tomato plant against aphids. The multitrophic system included the plant Solanum lycopersicum ('San Marzano nano'), the root-associated biocontrol fungus Trichoderma longibrachiatum strain MK1, the aphid Macrosiphum euphorbiae (a tomato pest), the aphid parasitoid Aphidius ervi, and the aphid predator Macrolophus pygmaeus. Laboratory bioassays were performed to assess the effect of T. longibrachiatum MK1, interacting with the tomato plant, on quantity and quality of volatile organic compounds (VOC) released by tomato plant, aphid development and reproduction, parasitoid behavior, and predator behavior and development. When compared with the uncolonized controls, plants whose roots were colonized by T. longibrachiatum MK1 showed quantitative differences in the release of specific VOC, better aphid population growth indices, a higher attractiveness toward the aphid parasitoid and the aphid predator, and a quicker development of aphid predator. These findings support the development of novel strategies of integrated control of aphid pests. The species-specific or strain-specific characteristics of these below ground-above ground interactions remain to be assessed. © 2013 The American Phytopathological Society.

The blend of volatile compounds emitted by tomato plants (Solanum lycopersicum) infested with the potato aphid (Macrosiphum euphorbiae) has been studied comparatively with undamaged plants and aphids themselves. Aphid-infested plants were significantly more attractive towards Aphidius ervi than undamaged plants and aphids themselves. Oriented response towards host-damaged plant, from which aphids were removed just before running the bioassay, did not differ from that recorded for infested plants. Collection of the volatiles and analysis by gas chromatography revealed only quantitative differences between uninfested and aphid-infested plants. Nine compounds, agr-pinene, (Z)-3-hexen-1-ol, agr-phellandrene, limonene, (E)-β-ocimene, p-cymene, methyl salicylate, (E)-β-caryophyllene and an unknown compound, were emitted at higher levels from aphid-infested plants than from undamaged control plants, whilst no differences were noted for hexanal, 6-methyl-5-hepten-2-one, and humulene (=agr-caryophyllene). Synthetic standards of these compounds were tested in wind tunnel bioassays and all elicited a significant increase in oriented flight and landings on the target by the aphid parasitoid Aphidius ervi. (E)-β-caryophyllene resulted the most attractive towards female wasps. These results corroborate the hypothesis that the volatiles produced by the plant in response to aphid attack derive from both jasmonic and salicylic acid pathways, and are exploited by A. ervi as olfactory cues to locate its hosts.

We have set up an analytical cell fractionation procedure to dissect, by a non-morphological method, the anterograde transport of proteins from endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells. Using this procedure after pulse-chase labelling of cells expressing human CD8 glycoprotein, we obtained results that: (1) support the view that the intermediate compartment is a distinct station in the export from the endoplasmic reticulum to the Golgi complex; and (2) strongly suggests that the O -glycosylation process starts after the intermediate compartment, presumably in the cis -Golgi complex.