Ilenia Siciliano

Insights on the Impact of Arbuscular Mycorrhizal Symbiosis on Tomato Tolerance to Water Stress

Arbuscular mycorrhizal symbiosis can improve tolerance to severe water stress conditions in tomato plants. Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most important crop species, are usually considered biofertilizers, whose exploitation could represent a promising avenue for the development in the future of a more sustainable next-generation agriculture. The best understood function in symbiosis is an improvement in plant mineral nutrient acquisition, as exchange for carbon compounds derived from the photosynthetic process: this can enhance host growth and tolerance to environmental stresses, such as water stress (WS). However, physiological and molecular mechanisms occurring in arbuscular mycorrhiza-colonized plants and directly involved in the mitigation of WS effects need to be further investigated. The main goal of this work is to verify the potential impact of AM symbiosis on the plant response to WS. To this aim, the effect of two AM fungi (Funneliformis mosseae and Rhizophagus intraradices) on tomato (Solanum lycopersicum) under the WS condition was studied. A combined approach, involving ecophysiological, morphometric, biochemical, and molecular analyses, has been used to highlight the mechanisms involved in plant response to WS during AM symbiosis. Gene expression analyses focused on a set of target genes putatively involved in the plant response to drought, and in parallel, we considered the expression changes induced by the imposed stress on a group of fungal genes playing a key role in the water-transport process. Taken together, the results show that AM symbiosis positively affects the tolerance to WS in tomato, with a different plant response depending on the AM fungi species involved.

Jasmonic Acid, Abscisic Acid, and Salicylic Acid Are Involved in the Phytoalexin Responses of Rice to Fusarium fujikuroi, a High Gibberellin Producer Pathogen.

Fusarium fujikuroi, the causal agent of bakanae disease, is the main seedborne pathogen on rice. To understand the basis of rice resistance, a quantitative method to simultaneously detect phytohormones and phytoalexins was developed by using HPLC-MS/MS. With this method dynamic profiles and possible interactions of defense-related phytohormones and phytoalexins were investigated on two rice cultivars, inoculated or not with F. fujikuroi. In the resistant cultivar Selenio, the presence of pathogen induced high production of phytoalexins, mainly sakuranetin, and symptoms of bakanae were not observed. On the contrary, in the susceptible genotype Dorella, the pathogen induced the production of gibberellin and abscisic acid and inhibited jasmonic acid production, phytoalexins were very low, and bakanae symptoms were observed. The results suggested that a wide range of secondary metabolites are involved in plant defense against pathogens and phytoalexin synthesis could be an important factor for rice resistance against bakanae disease.

Comparative transcriptome profiling of resistant and susceptible rice genotypes in response to the seedborne pathogen Fusarium fujikuroi

BackgroundFusarium fujikuroi is the causal agent of bakanae, the most significant seed-borne disease of rice. Molecular mechanisms regulating defence responses of rice towards this fungus are not yet fully known. To identify transcriptional mechanisms underpinning rice resistance, a RNA-seq comparative transcriptome profiling was conducted on infected seedlings of selected rice genotypes at one and three weeks post germination (wpg).ResultsTwelve rice genotypes were screened against bakanae disease leading to the identification of Selenio and Dorella as the most resistant and susceptible cultivars, respectively. Transcriptional changes were more appreciable at 3 wpg, suggesting that this infection stage is essential to study the resistance mechanisms: 3,119 DEGs were found in Selenio and 5,095 in Dorella. PR1, germin-like proteins, glycoside hydrolases, MAP kinases, and WRKY transcriptional factors were up-regulated in the resistant genotype upon infection with F. fujikuroi. Up-regulation of chitinases and down-regulation of MAP kinases and WRKY transcriptional factors were observed in the susceptible genotype. Gene ontology (GO) enrichment analyses detected in Selenio GO terms specific to response to F. fujikuroi: ‘response to chitin’, ‘jasmonic acid biosynthetic process’, and ‘plant-type hypersensitive response’, while Dorella activated different mechanisms, such as ‘response to salicylic acid stimulus’ and ‘gibberellin metabolic process’, which was in agreement with the production of gibberellin A3 in Dorella plants.ConclusionsRNA-seq profiling was performed for the first time to analyse response of rice to F. fujikuroi infection. Our findings allowed the identification of genes activated in one- and three- week-old rice seedlings of two genotypes infected with F. fujikuroi. Furthermore, we found the pathways involved in bakanae resistance, such as response to chitin, JA-dependent signalling and hypersensitive response. Collectively, this provides important information to elucidate the molecular and cellular processes occurring in rice during F. fujikuroi infection and to develop bakanae resistant rice germplasm.

Mycotoxin Production in Liquid Culture and on Plants Infected with Alternaria spp. Isolated from Rocket and Cabbage

Fungi belonging to the genus Alternaria are common pathogens of fruit and vegetables with some species able to produce secondary metabolites dangerous to human health. Twenty-eight Alternaria isolates from rocket and cabbage were investigated for their mycotoxin production. Five different Alternaria toxins were extracted from synthetic liquid media and from plant material (cabbage, cultivated rocket, cauliflower). A modified Czapek-Dox medium was used for the in vitro assay. Under these conditions, more than 80% of the isolates showed the ability to produce at least one mycotoxin, generally with higher levels for tenuazonic acid. However, the same isolates analyzed in vivo seemed to lose their ability to produce tenuazonic acid. For the other mycotoxins; alternariol, alternariol monomethyl ether, altenuene and tentoxin a good correlation between in vitro and in vivo production was observed. In vitro assay is a useful tool to predict the possible mycotoxin contamination under field and greenhouse conditions.

Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins

Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N2, 0.1% O2 and 1% O2, 21% O2), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB1 of over 70% was obtained. Aflatoxins B1 and G1 were more sensitive to plasma treatments compared to aflatoxins B2 and G2, respectively. Under plasma treatment, aflatoxin B1 was more sensitive compared to aflatoxin G1. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics.

Effect of Elevated Atmospheric CO2 and Temperature on the Disease Severity of Rocket Plants Caused by Fusarium Wilt under Phytotron Conditions

The severity of F. oxysporum f.sp. conglutinans on rocket plants grown under simulated climate change conditions has been studied. The rocket plants were cultivated on an infested substrate (4 log CFU g-1) and a non-infested substrate over three cycles. Pots were placed in six phytotrons in order to simulate different environmental conditions: 1) 400–450 ppm CO2, 18–22°C; 2) 800–850 ppm CO2, 18–22°C; 3) 400–450 ppm CO2, 22–26°C, 4) 800–850 ppm CO2, 22–26°C, 5) 400–450 ppm CO2, 26–30°C; 6) 800–850 ppm CO2, 26–30°C. Substrates from the infested and control samples were collected from each phytotron at 0, 60 and 120 days after transplanting. The disease index, microbial abundance, leaf physiological performances, root exudates and variability in the fungal profiles were monitored. The disease index was found to be significantly influenced by higher levels of temperature and CO2. Plate counts showed that fungal and bacterial development was not affected by the different CO2 and temperature levels, but a significant decreasing trend was observed from 0 up to 120 days. Conversely, the F. oxysporum f.sp. conglutinans plate counts did not show any significantly decrease from 0 up to 120 days. The fungal profiles, evaluated by means of polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), showed a relationship to temperature and CO2 on fungal diversity profiles. Different exudation patterns were observed when the controls and infested plants were compared, and it was found that both CO2 and temperature can influence the release of compounds from the roots of rocket plants. In short, the results show that global climate changes could influence disease incidence, probably through plant-mediated effects, caused by soilborne pathogens.

Characterization of Citrus-Associated Alternaria Species in Mediterranean Areas

Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. Recently, outbreaks in Mediterranean areas related to susceptible cultivars, refocused attention on the disease. Twenty representatives were selected from a collection of 180 isolates of Alternaria spp. from citrus leaves and fruit. They were characterized along with reference strains of Alternaria spp. Micro- and macroscopic characteristics separated most Alternaria isolates into six morphotypes referable to A. alternata (5) and A. arborescens (1). Phylogenetic analyses, based on endopolygalacturonase (endopg) and internal transcribed spacer (ITS), confirmed this finding. Moreover, a five-gene phylogeny including two anonymous genomics regions (OPA 1–3 and OPA 2–1), and the beta-tubulin gene (ß-tub), produced a further clustering of A. alternata into three clades. This analysis suggested the existence of intra-species molecular variability. Investigated isolates showed different levels of virulence on leaves and fruit. In particular, the pathogenicity on fruit seemed to be correlated with the tissue of isolation and the clade. The toxigenic behavior of Alternaria isolates was also investigated, with tenuazonic acid (TeA) being the most abundant mycotoxin (0.2–20 mg/L). Isolates also synthesized the mycotoxins alternariol (AOH), its derivate alternariol monomethyl ether (AME), and altenuene (ALT), although to a lesser extent. AME production significantly varied among the six morphotypes. The expression of pksJ/pksH, biosynthetic genes of AOH/AME, was not correlated with actual toxin production, but it was significantly different between the two genotypes and among the four clades. Finally, ten isolates proved to express the biosynthetic genes of ACTT1 phytotoxin, and thus to be included in the Alternaria pathotype tangerine. A significant correlation between pathogenicity on leaves and ACTT1 gene expression was recorded. The latter was significantly dependent on geographical origin. The widespread occurrence of Alternaria spp. on citrus fruit and their ability to produce mycotoxins might represent a serious concern for producers and consumers.

Effect of different temperatures and CO2 levels on Alternaria toxins produced on cultivated rocket, cabbage and cauliflower

Over the last 100 years, the global mean temperature has increased and has influenced several key factors that affect the occurrence and severity of fungal diseases. The effect of an increase in CO2 concentration and temperature on disease caused by four Alternaria strains and their mycotoxin production on cultivated rocket, cabbage and cauliflower plants has been investigated in this study. Six different temperature and CO2 combinations were considered: (1) 400-450 ppm CO2, 14-18 °C; (2) 800-850 ppm CO2, 14-18 °C; (3) 400-450 ppm CO2, 18-22 °C; (4) 800-850 ppm CO2, 18-22 °C; (5) 400-450 ppm CO2, 22-26 °C; and (6) 800-850 ppm CO2, 22-26 °C. Higher levels of CO2 and temperature have been found to significantly influence the disease index of the infected plants. In fact, the disease index was significantly increased at 22-26 °C and 800-850 ppm of CO2 for all of the host plants. Tenuazonic acid (TeA), alternariol, alternariol monomethyl ether and tentoxin were analysed for each climate condition using HPLC-M...

Static Hot Air and Infrared Rays Roasting are Efficient Methods for Aflatoxin Decontamination on Hazelnuts

Aflatoxins are a group of secondary metabolites produced by members of Aspergillus Section Flavi that are dangerous to humans and animals. Nuts can be potentially contaminated with aflatoxins, often over the legal threshold. Food processes, including roasting, may have different effects on mycotoxins, and high temperatures have proven to be very effective in the reduction of mycotoxins. In this work, two different roasting methods—traditional static hot air roasting and infra-red rays roasting—were applied and compared for the detoxification of hazelnuts from Italy and Turkey. At the temperature of 140 °C for 40 min of exposure, detoxification was effective for both roasting techniques. Residual aflatoxins after infra-red rays treatments were lower compared to static hot air roasting. On Italian hazelnuts, residual aflatoxins were lower than 5%, while for Turkish hazelnuts they were lower than 15% after 40 min of exposure to an infra-red rays roaster. After roasting, the perisperm was detached from the nuts and analyzed for aflatoxin contents. Residual aflatoxins in the perisperm ranged from 80% up to 100%. After roasting, the lipid profile and the nutritional quality of hazelnuts were not affected. Fatty acid methyl esters analyses showed a similar composition for Italian and Turkish hazelnuts.

Grapevine Grafting: Scion Transcript Profiling and Defense-Related Metabolites Induced by Rootstocks

Rootstocks are among the main factors that influence grape development as well as fruit and wine composition. In this work, rootstock/scion interactions were studied using transcriptomic and metabolic approaches on leaves of the “Gaglioppo” variety, grafted onto 13 different rootstocks growing in the same vineyard. The whole leaf transcriptome of “Gaglioppo” grafted onto five selected rootstocks showed high variability in gene expression. In particular, significant modulation of transcripts linked to primary and secondary metabolism was observed. Interestingly, genes and metabolites involved in defense responses (e.g., stilbenes and defense genes) were strongly activated particularly in the GAG-41B combination, characterized in addition by the down-regulation of abscisic acid (ABA) metabolism. On the contrary, the leaves of “Gaglioppo” grafted onto 1103 Paulsen showed an opposite regulations of those transcripts and metabolites, together with the greater sensitivity to downy mildew in a preliminary in vitro assay. This study carried out an extensive transcriptomic analysis of rootstock effects on scion leaves, helping to unravel this complex interaction, and suggesting an interesting correlation among constitutive stilbenes, ABA compound, and disease susceptibility to a fungal pathogen.