A cDNA of 312 bp, similar to polygalacturonase-inhibiting proteins(PGIPs), was isolated by cDNA-amplified fragment lengthpolymorphism (cDNA-AFLP) from pea roots infected with thecyst nematode Heterodera goettingiana. The deduced aminoacid sequence obtained from the complete Pspgip1 codingsequence was very similar to PGIPs described from several otherplant species, and was identical in both MG103738 and Progress9 genotypes, resistant and susceptible to H. goettingiana, respectively.Reverse transcription-polymerase chain reaction (RT-PCR)expression analysis revealed the differential regulation of thePspgip1 gene in the two genotypes in response to wounding andnematode challenge. Mechanical wounding induced Pspgip1expression in MG103738 within 8 h, but this response wasdelayed in Progress 9. In contrast, the response to nematodeinfection was more complex. The transcription of Pspgip1 wastriggered rapidly in both genotypes, but the expression levelreturned to levels observed in uninfected plants more quickly insusceptible than in resistant roots. In addition, in situ hybridizationshowed that Pspgip1 was expressed in the cortical cellsdamaged as a result of nematode invasion in both genotypes.However, it was specifically localized in the cells bordering thenematode-induced syncytia in resistant roots. This suggests arole for this gene in counteracting nematode establishmentinside the root.

Plant endoparasitic nematodes induce the formation of their feeding cells by injecting effectors from the esophageal glands into root cells. Although vascular cylinder cells seem to be involved in the formation of root-knot nematode (RKN) feeding structures, molecular evidence is scarce. We address the role during gall development of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), a key component of the auxin pathway leading to the divisions in the xylem pole pericycle (XPP) for lateral root (LR) formation.Arabidopsis T-DNA tagged J0192 and J0121 XPP marker lines, LBD16 and DR5::GUS promoter lines, and isolated J0192 protoplasts were assayed for nematode-dependent gene expression. Infection tests in LBD16 knock-out lines were used for functional analysis.J0192 and J0121 lines were activated in early developing galls and giant cells (GCs), resembling the pattern of the G2/M-transition specific ProCycB1;1:CycB1;1(NT)-GUS line. LBD16 was regulated by auxins in galls as in LRs, and induced by RKN secretions. LBD16 loss of function mutants and a transgenic line with defective XPP cells showed a significantly reduced infection rate.The results show that genes expressed in the dividing XPP, particularly LBD16, are important for gall formation, as they are for LR development.

Plant endoparasitic nematodes induce the formation of their feeding cells by injecting effectors from the esophageal glands into root cells. Although vascular cylinder cells seem to be involved in the formation of root-knot nematode (RKN) feeding structures, molecular evidence is scarce. We address the role during gall development of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), a key component of the auxin pathway leading to the divisions in the xylem pole pericycle (XPP) for lateral root (LR) formation. Arabidopsis T-DNA tagged J0192 and J0121 XPP marker lines, LBD16 and DR5::GUS promoter lines, and isolated J0192 protoplasts were assayed for nematode-dependent gene expression. Infection tests in LBD16 knock-out lines were used for functional analysis. J0192 and J0121 lines were activated in early developing galls and giant cells (GCs), resembling the pattern of the G2/M transition specific ProCycB1;1:CycB1;1(NT)-GUS line. LBD16 was regulated by auxins in galls as in LRs, and induced by RKN secretions. LBD16 loss of function mutants and a transgenic line with defective XPP cells showed a significantly reduced infection rate. The results show that genes expressed in the dividing XPP, particularly LBD16, are important for gall formation, as they are for LR development.

Main conclusion BTH application is effective in root-knot nematode-tomato interaction in a way that involves a delay in the formation of nematode feeding site and triggers molecular responses at several levels. The compatible interaction between root-knot nematodes and their hosts requires the nematode to overcome plant defense systems so that a sophisticated permanent feeding site (giant cells) can be produced within the host roots. It has been suggested that activators of plant defenses may provide a novel management strategy for controlling root-knot nematodes but little is known about the molecular basis by which these elicitors operate. The role of pre-treatment with Benzothiadiazole (BTH), a salicylic acid analog, in inducing resistance against Meloidogyne incognita infection was investigated in tomato roots. A decrease in galling in roots and feeding site numbers was observed following BTH treatment. Histological investigations showed a delay in formation of feeding sites in treated plants. BTH-treated galls had higher H2O2 production, lignin accumulation, and increased peroxidase activity than untreated galls. The expression of two tomato genes, Tap1 and Tap2, coding for anionic peroxidases, was examined by qRT-PCR and in situ hybridization in response to BTH. Tap1 was induced at all infection points, reaching the highest level at 15 dpi. Tap2 expression, although slightly delayed in untreated galls, increased during infection in both treated and untreated galls. The expression of Tap1 and Tap2 was observed in giant cells of untreated roots, whereas the transcripts were localized in both giant cells and in parenchyma cells surrounding the developing feeding sites in treated plants. These results show that BTH applied to tomato plants makes them more resistant to infection by nematodes, which become less effective in overcoming root defense pathway.

Benzothiadiazole (BTH) acts as a priming agent in plant defence leading to a reduction in penetration and development of the root-knot nematode Meloidogyne incognita in susceptible tomato roots. Changes in lignin biosynthesis in the susceptible tomato cv. Roma following nematode infection and/or BTH treatment were investigated in comparison to the resistant cv. Rossol. Both untreated and BTH-treated susceptible infected roots (galls) showed an increased level of expression of lignin synthesis-related genes (PAL, C4H, HCT and F5H) at early times during infection (2-4 days post inoculation). Peroxidase (soluble and cell-wall bound, POX) enzyme activities increased after inoculation with M. incognita and the priming effect of BTH treatment was evident at later stages of infection (7 days post inoculation). As expected, the induction of PAL and POXs and lignin synthesis-related genes was faster and greater in resistant roots after infection. Histochemical analysis revealed accumulation of higher lignin levels at later infection stages in BTH-treated galls compared to untreated ones. Furthermore, the monomer composition of lignin indicated a different composition in guaiacyl (G) and syringyl (S) units in BTH-treated galls compared to untreated galls. The increase in G units made G/S ratio similar to that in the resistant genotype. Overall, lignin played a critical role in tomato defence to M. incognita in response to BTH.

Biocontrol effects of arbuscular mycorrhizal fungi against nematodes have been reported in various plants. Literature data suggest that mycorrhizal symbiosis affects plant-water relationships as well. Moreover, it is well established that water deficit and infection with plant parasitic nematodes represent two environmental stresses with interacting effects under field conditions. Few data are available on the effect of combined mycorrhizae and water stress on the development of nematode feeding sites. We studied the impact of Rizophagus intraradices symbiosis on Meloidogyne incognita and tomato (cv San Marzano nano) interaction, with or without water stress. Plants inoculated or not with R. intraradices, maintained in growth chamber at 25°C, were exposed to mild water stress and subsequently infected with J2s of M. incognita. Galls hand-dissected at 7 and 14 days were processed for light microscopy observations. The analysis performed on cross sections of galls i) with or without mycorrhizae, ii) with water stress and iii) with mycorrhizae and water stress, showed changes in the morphology of galls and nematode feeding sites, affecting density and dimensions of nuclei. The symbiosis with R. intraradices and water stress hampered development and structure of giant cells, showing an effect on the modulation of host plant metabolism. NGS-based analysis of galls transcriptome is under study, to unravel the molecular pathways involved in this multiple interaction. Research partially funded by CNR, Progetto Premiale Aqua.

Ozone is widely used as a disinfectant, and ozonated water has been known to confer some protection of plants against several biotic stresses. By applying four foliar spray treatments of ozonated water (10 ppm ozone) on tomato seedlings, i.e. two pre- and two post-inoculation with Tomato spotted wilt virus (TSWV), we observed reduction of disease incidence and severity by 20%, as well as a virus titrrreduction by 80% at 19 days post-inoculation. The same treatments also reduced the number of galls induced by root knot nematode (RKN; Meloidogyne incognita) by 29%. Soil drenching with ozonated water for four consecutive days before inoculation reduced RKN gall formation by 60%, but not TSWV infection. Overall, in mock-inoculated plants, foliar sprays induced PR1b1 expression in leaves, though other salicylate- (PAL and PR-5x) or jasmonate-dependent genes (LoxD, AOS and PinI) were substantially unaffected. Soil drenching promptly enhancedtranscription of PAL and PR1b1 in roots and leaves, down-regulated PR-5x and did not affect expression of LoxD and AOS. PinI was significantly down-regulated only in leaves. The impact of ozonated water applications on the expression of these genes did not correlate with that of benzothiadiazole, a known inducer of systemic acquired resistance. This demonstrates that ozonated water may protect tomato fromtwo very different biotic stresses, especially when applied at the sites of their infection, and modulates salicylate and jasmonate pathways differently from benzothiadiazole. This research was supported by the Fondazione Cassa di Risparmio di Puglia, Italy, within the Project 'Risposte di difesa contro nematodi e virus indotte da trattamenti di ozono in pomodoro'.

Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound gamma-tubulin ring complexes. Here we investigate the requirement of gamma-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that gamma-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis c-tubulin (TUBG1 and TUBG2) and two gamma-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and gamma-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both gamma-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the gamma-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

Annually significant tomato yield losses are caused by climate changes and pests and diseases attacks. Root-knot nematodes (Meloidogyne spp.) are the most damaging pests on the base of their wide distribution in tropic and sub-tropic climates and their wide host range. They modify host root tissue using effector proteins to create feeding sites as their source of nutrition. To develop alternative control strategies to nematode infections, we have considered aqueous ozone treatments (AOT) as a possible tool. Although gaseous ozone is phytotoxic, previous studies demonstrated that ozone in aqueous phase is effective to contain pests. The purposes of this study were to confirm the impact of AOT on tomato-M. incognita interaction and to analyse its role in plant defence response. Ozonated water was produced in situ by an ozone generator at 10 ppm and directly applied to tomato roots. Treatments were performed daily (10 ml/pot) for 4 days as soil drench on 14 days old tomato plants maintained in a growth cabinet (25 ± 2 °C). Nematode infection control by ozone treatments was set up by infecting untreated and treated plants with J2s of M. incognita. A number of plants were used 2, 4 and 7 days after nematode infection for biochemical and molecular analysis. Other plants were transferred in a glasshouse at 25 ± 2 °C and after 60 days they were uprooted and dry shoot and root fresh weights recorded. Root gall index, eggs and J2s/g root, total nematode population density and reproduction rate were evaluated. AOT significantly decreased severity of root gall index and soil nematode population in comparison to untreated plants. Tomato sensitivity to AOT was verified as reactive oxygen species production (ROS) in both treated and untreated root galls and compared with uninfected treated and untreated roots. A higher significant ROS production was observed in ozone treated uninfected roots in comparison to treated and untreated infected roots. As antioxidant mechanisms play an important role in the response of plant to the combination of abiotic and biotic stresses, the effect of AOT on the expression of different genes involved in ROS scavenging, such as CAT, SOD and APX, was also evaluated. AOT by altering gene expression, ROS production and inducing a non specific defense response can be considered a useful tool to contain nematode infection.Aknowledgement: This work was supported with funds provided by the National Research Council for the project "Innovazione e Sviluppo del Mezzogiorno - Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare".

Biological processes using microorganisms for nanoparticle synthesis are appealing as eco-friendly nanofac-tories. The response of the photosynthetic bacterium Rhodobacter sphaeroides to gold exposure and its reducingcapability of Au(III) to produce stable gold nanoparticles (AuNPs), using metabolically active bacteria andquiescent biomass, is reported in this study.In the former case, bacterial cells were grown in presence of gold chloride at physiological pH. Gold exposurewas found to cause a significant increase of the lag-phase duration at concentrations higher than 10 ?M, sug-gesting the involvement of a resistance mechanism activated by Au(III). Transmission Electron Microscopy(TEM) and Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDS) analysis of bac-terial cells confirmed the extracellular formation of AuNPs.Further studies were carried out on metabolically quiescent biomass incubated with gold chloride solution.The biosynthesized AuNPs were spherical in shape with an average size of 10 ± 3 nm, as analysed byTransmission Electron Microscopy (TEM). The nanoparticles were hydrophilic and stable against aggregation forseveral months.In order to identify the functional groups responsible for the reduction and stabilization of nanoparticles,AuNPs were analysed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, X-ray Photoelectron Spectroscopy (XPS), X-ray Fluorescence Spectrometry (XRF) and X-ray AbsorptionSpectroscopy (XAS) measurements. The obtained results indicate that gold ions bind to functional groups of cellmembrane and are subsequently reduced by reducing sugars to gold nanoparticles and capped by a protein/peptide coat.Gold nanoparticles demonstrated to be efficient homogeneous catalysts in the degradation of nitroaromaticcompounds.

Sedentary endoparasitic root-knot nematodes (Meloidogyne spp.) are one of the most damaging pest in global agriculture. Various approaches, most focusing on the introduction of resistance genes in plants, have been undertaken to control these pathogens. Recently, plant activators provide an appealing management option by stimulating active plant defence mechanisms. One of the most interesting activators is benzo (1,2,3) thiodiazole-7-carbothioic acid S-methyl ester (BTH), which induces a broad spectrum, long lasting, and systemic immunity against different pathogens. The effect of the BTH pre-treatment in the compatible interaction tomato-Meloidogyne incognita was investigated. We found that BTH significantly reduces nematode reproduction and affects morphology of nematode feeding sites, thus enhancing resistance against infection.

Few studies have been carried out on the effect of ozonated water (O3wat) on the oxidative stress of root systems and, in particular,in combination with biotic stress. The aim of this study was to determine whether aqueous ozone is effective in the control of root-knot nematode (RKN) infection and to investigate the concomitant changes in the basal defence system. A tomato cultivar susceptible to Meloidogyne incognita was treated with O3wat as a soil drench. No negative effects were seen following ozone application in comparison with the control under the exposure conditions used. The treatment reduced significantly the nematode infection rate and induced changes in the morphology of nematode feeding sites, some of which were characterized by visible symptoms of senescence. The antioxidant response, as well as parameters of oxidative damage, were examined in untreated and Owat-treated galls at 2, 4 and 7 days after inoculation and compared with uninfected roots. High levels of reactive oxygen species (ROS), H32O and malondialdehyde were generated in galls in response to combined abiotic and biotic stresses. Throughout the experimental period, the activities and relative transcript levels of the antioxidant enzymes catalase, superoxide dismutase and ascorbate peroxidase produced different responses when exposed to ozone treatment and/or infection. The results demonstrate how O32wat protects tomato against the RKN M. incognita through the modulation of basal defence mechanisms.

Aqueous ozone treatments were applied to tomato plants (cv. Roma VF) before and after Meloidogyne incognita infection in controlled growth conditions. Ozonated water was produced in situ by an ozone generator at 8 ppm. Treatments were applied for 3 and 4 days as soil drench at the rate of 10 ml/pot (50 ml). Untreated plants were used as control. The sensitivity to aqueous ozone treatments was verified by the production of reactive oxygen species (ROS) in roots. A significant ROS production was observed after two days of ozone treatments in comparison to the untreated plants, without adversely influencing the growth of the treated plants. Visual examination of the root systems revealed no symptomatic evidence of root browning or other damages indicative of oxidative stress. Tomato plants infected with M. incognita before and after ozone treatments were grown in a glasshouse at 25 ± 2°C. After 60 days the plants were uprooted, and dry shoot and root fresh weights were recorded. Root gall index, eggs and juveniles/g root, total nematode population density and reproduction rates were evaluated. Root gall index was assessed according to a 0-10 scale, whereas nematodes were extracted from roots and soil by centrifugation and the Coolen's method, respectively. Reproduction rate was calculated by the ratio between final and initial nematode population density (r =Pf/Pi). Data from the experiments were subjected to ANOVA and means compared by Least Significant Difference's test. Root gall index, eggs and juveniles/g root, total nematode population density and the reproduction rate were significantly reduced in both ozone treatments (3 and 4 days of treatments), either before or after nematode inoculation, in comparison to the untreated control.

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. To investigate the key components involved in oxidative and nitrosative metabolism, experiments were carried out at different infection times by using a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita. We analyzed the superoxide radical (O2.-) production, hydrogen peroxide (H2O2) content, and nitric oxide synthase (NOS)-like, and nitrate reductase (NR) activities, as potential sources of NO. A rapid NO accumulation and ROS production were differently linked to incompatible and compatible tomato-nematode interactions. NOS-like arginine-dependent rather than NR was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H2O2 cooperating in triggering hypersensitive cell death. The results obtained from this studies reveal a defence mechanism that has not been previously described in tomato-nematode interaction and provide new insight into the complex regulation of ROS and NO metabolism by avr- and vir- RKN pathotypes in their hosts roots.

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomatonematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatibleinteraction.We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H2O2 cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.