Salicylic acid foliar application meliorates Portulaca oleraceae L. growth responses under Pb and Ni over-availability while keeping reliable phytoremediation potential.

The efficacy of SA foliar use on Pb and Ni-induced stress tolerance and phytoremediation potential by Portulaca oleraceae L. were assayed as a factorial trial based on a completely randomized design with four repetitions. The factors included; SA foliar application (0 and 100 µM) and HMs application of Pb [0, 150, and 225 mg kg-1 soil Lead (II) nitrate] and Ni [0, 220, and 330 mg kg-1 soil Nickel (II) nitrate]. Plant height, stem diameter, shoot and root fresh and dry weight, photosynthetic pigments, total soluble proteins, palmitic acid, stearic acid, arachidic acid, and some macro- and micro-elements contents were reduced facing the HMs stress, but SA foliar application ameliorated these traits. HMs stress increased malondialdehyde content, total antioxidant activity, total flavonoids, phenolics, and linolenic acid content, while SA foliar application declined the mentioned parameters. Moreover, shoot and root Pb and Ni content enhanced in the purslane plants supplemented by SA under the HMs stress. The results propose SA foliar application as a reliable methodology to recover purslane growth characters and fatty acid profiles in the soil contaminated with the HMs. The idea is that SA would be potentially effective in alleviating HMs contamination while keeping reasonable phytoremediation potential.

There is no information available in previous literature about the impact of Pb and Ni on the phytochemical profile of oil in purslane. Therefore, in this report, we evaluated the purslane plant's growth and physiological responses and its seed oil's components in response to SA foliar application under conditions of Pb and Ni over-availability. Additionally, we examined the role of SA treatment in improving phytoremediation of Pb and Ni.

Transcriptomic profile of lettuce seedlings (Lactuca sativa) response to microalgae extracts used as biostimulant agents.

To reduce the use of chemical fertilizers and maximize agricultural yields, the use of microalgae extracts as biostimulants has recently attracted significant attention due to their favourable impact on both plant growth and their ability to induce tolerance towards environmental stressors. Lettuce (Lactuca sativa) is one of the most important fresh vegetables that often requires applications of chemical fertilizers to increase quality and productivity. Accordingly, the purpose of this study was to analyse the transcriptome reprogramming of lettuce (L. sativa) seedlings in response to either Chlorella vulgaris or Scenedesmus quadricauda extracts by applying an RNAseq approach. Differential gene expression analysis revealed that the core gene set that responded to microalgal treatments in a species-independent manner includes 1330 clusters, 1184 of which were down-regulated and 146 up-regulated, clearly suggesting that the repression of gene expression is the main effect of algal treatments. The deregulation of 7197 transcripts in the C. vulgaris treated seedlings compared to control samples (LsCv vs. LsCK) and 7118 transcripts in the S. quadricauda treated seedlings compared to control samples (LsSq vs. LsCK) were counted. Although the number of deregulated genes turned out to be similar between the algal treatments, the level of deregulation was higher in LsCv versus LsCK than in LsSq versus LsCK. In addition, 2439 deregulated transcripts were observed in the C. vulgaris treated seedlings compared to S. quadricauda treated samples (LsCv vs. LsSq comparison) suggesting that a specific transcriptomic profile was induced by the single algal extracts. 'Plant hormone signal transduction' category includes a very elevated number of DEGs, many of them specifically indicating that C. vulgaris actives both genes involved in the auxin biosynthesis and transduction pathways, whereas S. quadricauda up-regulates genes implicated in the cytokinin biosynthesis pathway. Finally, algal treatments induced the deregulation of genes encoding small hormone-like molecules that are known to act alone or by interacting with major plant hormones. In conclusion, this study offers the groundwork to draw up a list of putative gene targets with the aim of lettuce genetic improvement that will allow a limited or even null use of synthetic fertilizers and pesticides in the management of this crop.

Improvements in the biochemical responses and Pb and Ni phytoremediation of lavender (Lavandula angustifolia L.) plants through Funneliformis mosseae inoculation.

BACKGROUND: Heavy metals (HMs) phytoremediation is a well-recognized protocol to remove toxic elements from the soil. As known, arbuscular mycorrhizal fungi (AMF) enhance the plants' growth responses. The idea of the present study was to assay the response of lavender plants to HMs stress under AMF inoculation. We hypothesized that mycorrhiza will enhance the phytoremediation and simultaneously reduce the harmful effects of heavy HMs. So, lavender (Lavandula angustifolia L.) plants were inoculated with AMF (0 and 5 g Kg-1 soil) under Pb [150 and 225 mg kg-1 soil from Pb (NO3)2] and Ni [220 and 330 mg kg-1 soil from Ni (NO3)2] pollution, in the greenhouse conditions. The control treatment was plants not treated with AMF and HMs. Doing this, the root colonization, HMs uptake, enzymatic and non-enzymatic antioxidants pool, MDA, proline, total phenolics (TPC), flavonoids (TFC), anthocyanins, and essential oil (EO) components were evaluated. RESULTS: According to the findings, the AMF inoculation enhanced shoot and root Pb and Ni content, antioxidant enzymes activity, the total antioxidant activity by DPPH and FRAP methods, TPC, TFC, anthocyanins, and H2O2 content in the lavender plants subjected to Pb and Ni stress. Moreover, the highest (28.91%) and the least (15.81%) percentages of borneol were identified in the lavender plants subjected to AMF under 150 mg kg-1 of Pb and the control plants without AMF application, respectively. Furthermore, the top 1,8-cineole (12.75%) content was recorded in AMF-inoculated plants. CONCLUSIONS: The overall results verify that AMF inoculation can be a reliable methodology to enhance the phytoremediation of Pb and Ni by lavender plants while maintaining reliable growth potential. The treatments improved the main EO constituents content, especially under moderate HMs stress conditions. With more detailed studies, the results will be advisable for the extension section for the phytoremediation of polluted soils.

Effect of municipal biowaste derived biostimulant on nitrogen fate in the plant-soil system during lettuce cultivation.

A main concern of agriculture is to improve plant nutrient efficiency to enhance crop yield and quality, and at the same time to decrease the environmental impact caused by the lixiviation of excess N fertilizer application. The aim of this study was to evaluate the potential use of biopolymers (BPs), obtained by alkaline hydrolysis of the solid anaerobic digestate of municipal biowastes, in order to face up these main concerns of agriculture. The experimental trials involved the application of BPs (at 50 and 150 kg/ha) alone or mixed with different amounts (100%, 60% and 0%) of mineral fertilizer (MF). Three different controls were routinely included in the experimental trials (MF 100%, 60% and 0%). The effect of BPs on lettuce was evaluated by monitoring growth parameters (fresh and dry weights of shoot and root, nitrogen use efficiency), and the N-flux in plant-soil system, taking into account the nitrate leached due to over irrigation events. The activities of enzymes involved in the nitrogen uptake (nitrate reductase, glutamate synthase and glutamine synthase), and the nitrogen form accumulated in the plant tissues (total N, protein and NO3-) were evaluated. The results show that the application to the soil of 150 kg/ha BPs allows to increase lettuce growth and nitrogen use efficiency, trough stimulation of N-metabolism and accumulation of proteins, and hence to reduce the use of MF by 40%, thus decreasing the nitrate leaching. These findings suggest that the use of BPs as biostimulant greatly contributes to reduce the consumption of mineral fertilizers, and to mitigate the environmental impact caused by nutrients leaching, according to European common agricultural policy, that encourages R&D of new bioproducts for sustainable eco-friendly agriculture.

Effects of a Novel Gel Formulation of Dog Appeasing Pheromone (DAP) on Behavioral and Physiological Stress Responses in Dogs Undergoing Clinical Examination.

The veterinary visit is necessary for safeguarding the health of dogs, but it can be stressful and threaten both the welfare of the patient and the accuracy of the examination. This randomized, triple-blind, placebo-controlled, crossover study aims at evaluating how dog appeasing pheromone (DAP) in a novel gel formulation influences the behavioral and physiological stress responses of 28 dogs undergoing a standardized clinical examination, while staying in the waiting room (WR) and visited in the examination room (ER). Behavioral responses were studied through behavioral categories and subjective scales (WR and ER). Autonomic response considered heart rate (WR and ER), blood pressure (WR and ER), respiratory rate (ER), and rectal temperature (ER). Neuroendocrine response considered salivary cortisol (WR and ER). In the waiting room, the use of DAP was associated with a significant reduction of lip licking (p = 0.0189), an increase in panting (p = 0.0276), and a reduction close to significance (p = 0.0584) of low body postures. No significant differences were observed within the physiological responses. In the examination room, neither behavioral nor physiological differences were found.

An Easy and Cheap Kiwi-Based Preparation as Vegetable Milk Coagulant: Preliminary Study at the Laboratory Scale.

In the present study, a kiwifruit aqueous extract was developed and used as a coagulant enzyme in cheesemaking. In detail, polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the presence of actinidin, the kiwifruit enzyme involved in κ-casein hydrolysis, in different tissues (pulp, peel, and whole fruit) of ripe and unripe kiwifruits. Data revealed the presence of the enzyme both in the peel and in the pulp of the fruit. Although the aqueous extract obtained from the kiwifruit peel was able to hydrolyze semi-skimmed milk, it did not break down κ-casein. The aqueous extract obtained from the pulp showed a hydrolytic activity toward both κ-casein and semi-skimmed milk. The values for milk-clotting and proteolytic activity of the kiwifruit pulp extract were evaluated at different temperatures and pH parameters in order to obtain a high value of the MCA/PA ratio; we found that a temperature of 40 °C in combination with a pH value of 5.5 allowed us to obtain the best performance. In addition, the data revealed a higher hydrolytic activity of the enzymatic preparation from ripe kiwifruits than that from unripe ones, suggesting the use of the extract from pulp of ripe kiwifruits in the laboratory-scale cheesemaking. The data showed that 3% (v/v) of the ripe kiwifruit pulp extract determined a curd yield of 20.27%, comparable to chymosin yield. In conclusion, the extraction procedure for kiwifruit aqueous extract proposed in the present study was shown to be a fast, cheap, chemical-free, and ecofriendly technology as a plant coagulant for cheese manufacturing.

Plant Milk-Clotting Enzymes for Cheesemaking.

The reduced availability and the increasing prices of calf rennet, coupled to the growing global demand of cheese has led, worldwide, to explore alternative clotting enzymes, capable to replace traditional rennet, during the cheesemaking. In addition, religious factors and others related to the vegetarianism of some consumers, have led to alternative rennet substitutes. Nowadays, several plant-derived milk-clotting enzymes are available for cheesemaking technology. Many efforts have also been made to compare their effects on rheological and sensory properties of cheese to those arising from animal rennet. However, vegetable clotting enzymes are still partially suitable for cheesemaking, due to excessive proteolytic activity, which contribute to bitter flavor development. This review provides a literature overview of the most used vegetable clotting enzymes in cheese technology, classified according to their protease class. Finally, clotting and proteolytic activities are discussed in relation to their application on the different cheesemaking products.

Side effects of two citrus essential oil formulations on a generalist insect predator, plant and soil enzymatic activities.

The widespread use of chemical pesticides for crop protection, despite having contributed to ensure food security, have shown to exert negative impacts on the environment and on human health. In addition, the frequent emergence of resistance to pesticides and their adverse effects toward non-target organisms have generated the need to develop novel ecofriendly tools for pest control. Among these, plant essential oils (EOs) may play a central role in arthropod pest control. Recently, two formulations (Emulsion and PEG-nanoparticles) of three citrus EOs (lemon, mandarin and sweet orange) showed a promising potential against Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), a key tomato pest. Here, we evaluated the side effects of these experimental insecticides active substances toward (i) the generalist predator of several tomato pests, Nesidiocoris tenuis Reuter (Hemiptera: Miridae); (ii) the soil enzymatic activities (dehydrogenase activity, alkaline phosphomonoesterase, acid phosphomonoesterase and urease) and (iii) the tomato plant antioxidant enzymes (ascorbate peroxidase, catalase, superoxide dismutase and polyphenol oxidase). Among the tested formulations, mandarin EO-based insecticide presented a significant impact on the predator survival and reproduction. Conversely, all the tested compounds proved to be harmless for the soil enzymatic and the plant antioxidant activities. Overall, these results provide solid bases for the development of novel biopesticides for sustainable tomato crop protection.

Effect of Microalgal Extracts from Chlorella vulgaris and Scenedesmus quadricauda on Germination of Beta vulgaris Seeds.

Sugar beet (Beta vulgaris subsp. vulgaris) is a commercially important biennial root crop, providing about 20% of the world's annual sugar production. Seed quality is crucial for adequate plant growth and production. The productivity of sugar beet is often limited by heterogeneous germination in the field. In order to improve the sugar beet germination process, the effect of different concentrations of microalgal extracts from Chlorella vulgaris or Scenedesmus quadricauda was investigated by calculating several indices useful to evaluate the germination performance. Moreover, root morphological analysis was performed by using WinRHIZO software. B. vulgaris seeds were soaked with five different concentrations (from 0.1 to 10 mg Corg/L) of the microalgal extracts, considering the amount of organic carbon (Corg) in each extract. Our results show that these microalgal extracts exert a positive effect on sugar beet germination, by increasing efficiency and regularity of this critical process for B. vulgaris seeds. The best results, in terms of germination indices as well as root morphological traits, were reached by using C. vulgaris extract at the concentrations C2 (1 mg Corg/L) and C3 (2 mg Corg/L).

Biostimulant Effect and Biochemical Response in Lettuce Seedlings Treated with A Scenedesmus quadricauda Extract.

The use of natural biostimulants is becoming an attractive option in order to reduce the use of fertilizer and increase the yield of crops. In particular, algal extracts are suitable candidates as they positively affect plant physiology. Among crops, lettuce often requires the use of biostimulants to improve both the quality and quantity of production. The aim of this work is to investigate the potential use of a Scenedesmus quadricauda extract as a biostimulant in order to obtain sustainable cultivation and a reduction in the cost of chemical fertilizers in lettuce cultivation. Therefore, the effect of S. quadricauda extract on lettuce seedlings was explored by evaluating the physiological parameters, chlorophyll, carotenoid, and total protein contents as well as several plant enzymatic activities involved in primary and secondary metabolisms. The experiment was performed by growing plants on inert substrate (pumice) with a 16-h photoperiod, by carrying out two consecutive radical treatments, one week apart, using a concentration of the extract corresponding to 1 mg Corg L-1. Lettuce plants were sampled at 1, 4, and 7 days from the first treatment and 7 days from the second treatment. The results showed that the S. quadricauda extract positively affected the growth of lettuce seedlings, mainly acting at the shoot level, determining an increase in dry matter, chlorophylls, carotenoids, proteins, and influencing the activities of several enzymes involved in the primary metabolism.

Physiological and Biochemical Responses of Orange Trees to Different Deficit Irrigation Regimes.

The article presents the results of research consisting of the application of deficit irrigation (DI) criteria, combined with the adoption of micro-irrigation methods, on orange orchards (Citrus sinensis (L.) Osbeck) in Sicily (Italy) during the irrigation season of 2015. Regulated deficit irrigation (RDI, T3) and partial root-zone drying (PRD, T4) strategies were compared with full irrigation (T1) and sustained deficit irrigation (SDI, T2) treatments in terms of physiological, biochemical, and productive crop response. A geophysical survey (electrical resistivity tomography, ERT) was carried out to identify a link between the percentages of drying soil volume in T4 with leaves abscisic acid (ABA) signal. Results highlight that the orange trees physiological response to water stress conditions did not show particular differences among the different irrigation treatments, not inducing detrimental effects on crop production features. ABA levels in leaves were rather constant in all the treatments, except in T4 during late irrigation season. ERT technique identified that prolonged drying cycles during alternate PRD exposed more roots to severe soil drying, thus increasing leaf ABA accumulation.

Expression Profiling of Candidate Genes in Sugar Beet Leaves Treated with Leonardite-Based Biostimulant.

Leonardite-based biostimulants are a large class of compounds, including humic acid substances. Foliar application of biostimulants at field level improves plant growth, yield and quality through metabolic changes and stimulation of plant proton pumps. The present study aimed at identifying optimum dosage of BLACKJAK, a humic acid-based substance, which is able to modify genes involved in sugar beet growth. Thirty-three genes belonging to various biochemical pathway categories were tested in leaves of treated sugar beet (Beta vulgaris L.) samples to assess gene expression profiling in response to BLACKJAK. Seedlings of a diploid and multigerm variety were grown in plastic pots and sprayed with two dilutions of BLACKJAK (dilution 1:500-1.0 mg C L-1 and dilution 1:1000-0.5 mg C L-1). Leaf samples were collected after 24, 48, and 72 h treatment with BLACKJAK for each dilution. RNA was extracted and the quantification of gene expression was performed while using an OpenArray platform. Results of analysis of variance demonstrated that, 15 genes out of a total of 33 genes tested with OpenArray qPCR were significantly affected by treatment and exposure time. Analysis for annotation of gene products and pathways revealed that genes belonging to the mitochondrial respiratory pathways, nitrogen and hormone metabolisms, and nutrient uptake were up-regulated in the BLACKJAK treated samples. Among the up-regulated genes, Bv_PHT2;1 and Bv_GLN1 expression exerted a 2-fold change in 1:1000 and 1:500 BLACKJAK concentrations. Overall, the gene expression data in the BLACKJAK treated leaves demonstrated the induction of plant growth-related genes that were contributed almost to amino acid and nitrogen metabolism, plant defense system, and plant growth.

Molecular and Morphological Changes Induced by Leonardite-based Biostimulant in Beta vulgaris L.

Humic substances extracted from leonardite are widely considered to be bioactive compounds, influencing the whole-plant physiology and the crop yield. The aim of this work was to evaluate the effect of a new formulate based on leonardite in the early stage of growth of sugar beet (Beta vulgaris L.). A commercial preparation of leonardite (BLACKJAK) was characterized by ionomic analysis, solid-state 13C MAS NMR spectroscopy. Seedlings of sugar beet were grown in Hoagland's solution under controlled conditions. After five days of growth, an aliquot of the concentrated BLACKJAK was added to the solution to obtain a final dilution of 1:1000 (0.5 mg C L-1). The sugar beet response in the early stage of growth was determined by evaluating root morphological traits as well as the changes in the expression of 53 genes related to key morphophysiological processes. Root morphological traits, such as total root length, fine root length (average diameter < 0.5 mm), and number of root tips, were significantly (p < 0.001) increased in plants treated with BLACKJAK, compared to the untreated plants at all sampling times. At the molecular level, BLACKJAK treatment upregulated many of the evaluated genes. Moreover, both Real Time PCR and digital PCR showed that genes involved in hormonal response, such as PIN, ARF3, LOGL 10, GID1, and BRI1, were significantly (p < 0.05) upregulated by treatment with BLACKJAK. Our study provides essential information to understand the effect of a leonardite-based formulate on plant growth hormone metabolism, although the molecular and physiological basis for these complicated regulatory mechanisms deserve further investigations.

Characterization of Phytophthora infestans populations in northwestern Algeria during 2008-2014.

A total of 161 Phytophthora infestans isolates, collected from infected potato and tomato plants during 2008-2014, were characterized based on mating type, metalaxyl sensitivity and polymorphism at 12 simple sequence repeat (SSR) loci, in order to investigate the population of P. infestans in the north-west of Algeria, an emerging potato production region. The majority of isolates were of A2 mating type (112 isolates). A high percentage (89 %) of resistance to metalaxyl among isolates was detected. The metalaxyl resistant phenotype was present in both mating types with a higher percentage in A2 mating type isolates. SSR-based genotypic analysis of P. infestans population showed a low diversity. Genotype 13_A2 was the predominant in the population with a frequency of 67 % followed by 2_A1 (21 %) and 23_A1 (5 %). Genotype 23_A1 was detected only in tomato and potato isolates collected in 2013 and 2014.

Two previously unknown Phytophthora species associated with brown rot of Pomelo (Citrus grandis) fruits in Vietnam.

Two distinct Phytophthora taxa were found to be associated with brown rot of pomelo (Citrus grandis), a new disease of this ancestral Citrus species, in the Vinh Long province, Mekong River Delta area, southern Vietnam. On the basis of morphological characters and using the ITS1-5.8S-ITS2 region of the rDNA and the cytochrome oxidase subunit 1 (COI) as barcode genes, one of the two taxa was provisionally named as Phytophthora sp. prodigiosa, being closely related to but distinct from P. insolita, a species in Phytophthora Clade 9, while the other one, was closely related to but distinct from the Clade 2 species P. meadii and was informally designated as Phytophthora sp. mekongensis. Isolates of P. sp. prodigiosa and P. sp. mekongensis were also obtained from necrotic fibrous roots of Volkamer lemon (C. volkameriana) rootstocks grafted with 'King' mandarin (Citrus nobilis) and from trees of pomelo, respectively, in other provinces of the Mekong River Delta, indicating a widespread occurrence of both Phytophthora species in this citrus-growing area. Koch's postulates were fulfilled via pathogenicity tests on fruits of various Citrus species, including pomelo, grapefruit (Citrus x paradisi), sweet orange (Citrus x sinensis) and bergamot (Citrus x bergamia) as well as on the rootstock of 2-year-old trees of pomelo and sweet orange on 'Carrizo' citrange (C. sinensis 'Washington Navel' x Poncirus trifoliata). This is the first report of a Phytophthora species from Clade 2 other than P. citricola and P. citrophthora as causal agent of fruit brown rot of Citrus worldwide and the first report of P. insolita complex in Vietnam. Results indicate that likely Vietnam is still an unexplored reservoir of Phytophthora diversity.

Cadmium induces cadmium-tolerant gene expression in the filamentous fungus Trichoderma harzianum.

The filamentous fungus Trichoderma harzianum, strain IMI 393899, was able to grow in the presence of the heavy metals cadmium and mercury. The main objective of this research was to study the molecular mechanisms underlying the tolerance of the fungus T. harzianum to cadmium. The suppression subtractive hybridization (SSH) method was used for the characterization of the genes of T. harzianum implicated in cadmium tolerance compared with those expressed in the response to the stress induced by mercury. Finally, the effects of cadmium exposure were also validated by measuring the expression levels of the putative genes coding for a glucose transporter, a plasma membrane ATPase, a Cd(2+)/Zn(2+) transporter protein and a two-component system sensor histidine kinase YcbA, by real-time-PCR. By using the aforementioned SSH strategy, it was possible to identify 108 differentially expressed genes of the strain IMI 393899 of T. harzianum grown in a mineral substrate with the addition of cadmium. The expressed sequence tags identified by SSH technique were encoding different genes that may be involved in different biological processes, including those associated to primary and secondary metabolism, intracellular transport, transcription factors, cell defence, signal transduction, DNA metabolism, cell growth and protein synthesis. Finally, the results show that in the mechanism of tolerance to cadmium a possible signal transduction pathway could activate a Cd(2+)/Zn(2+) transporter protein and/or a plasma membrane ATPase that could be involved in the compartmentalization of cadmium inside the cell.

The glutathione S-transferase gene superfamily: an in silico approach to study the post translational regulation.

The use of plants to reclaim contaminated soils and groundwater, known as phytoremediation, is a promising biotechnological strategy which has gained a lot of attention in the last few years. Plants have evolved sophisticated detoxification systems against the toxin chemicals: following the uptake, the compounds are activated so that certain functional groups can conjugate hydrophilic molecules, such as thiols. The resulting conjugates are recognized by the tonoplast transporters and sequestered into the vacuoles. The xenobiotic conjugation with glutathione is mediated by enzymes which belong to the superfamily of glutathione S-transferases (GSTs) catalyzing the nucleophylic attack of the sulphur of glutathione on the electrophilic groups of the cytotoxic substrates therefore playing a crucial role in their degradation. This study was designed to identify the putative correlation between structural and functional characteristics of plant GST classes belonging to different plant species. Consequently, the protein sequences of the expressed GSTs have been retrieved from UniGene, classified and then analyzed in order to assess the evolutionary trend and to predict secondary structure. Moreover, the fingerprint analysis was performed with SCAN Prosite in the attempt to correlate meaningful signature profile and biological information. The results evidenced that all the soluble GSTs have a tendency to assume the α-helix secondary structure followed by random coil and ß-sheet. The fingerprint analysis revealed that specific signature profiles related mainly to protein phosphorylation are in the GST classes of all considered species thus suggesting that they might be subjected to reversible activation by phosphorylation-mediated regulation. This approach provides the knowledge of the relationship between presence of conserved signature profile and biological function in the view of future selection of GSTs which might be employed in either mutagenesis or genetic engineering studies.

Identification of differentially expressed genes in response to mercury I and II stress in Trichoderma harzianum.

Filamentous fungi are very promising organisms in both the control and the reduction of the amount of heavy metal released by human and industrial activities. In particular, Trichoderma harzianum demonstrated to be tolerant towards different heavy metals, such as mercury and cadmium, even though the mechanism underlying this tolerance is not fully understood. By using a particular strategy of the suppression subtractive hybridization technique, we were able to identify in the strain IMI 393899 of T. harzianum eight different genes up-regulated in the presence of mercury II with respect to cadmium. Among the genes identified, a possible role in the tolerance mechanism could be envisaged for hydrophobin, due to its ability to dissolve hydrophobic molecules into aqueous media. We also show that IMI 393899 grows at the same rate of control culture in the presence of mercury I and that all eight genes isolated were also up-regulated in this condition.

Expression of genes of Trichoderma harzianum in response to the presence of cadmium in the substrate.

Trichoderma harzianum is a very important fungus for the reduction of the amount of heavy metals resulting from agricultural and industrial activities. This filamentous fungus has been shown to be tolerant towards several heavy metals (e.g. Cd, Pb, Zn, Ni and Mn), but the mechanism underlying this tolerance is not entirely understood. In this study, we confirmed the ability of T. harzianum to grow in the presence of cadmium and observed a significant stimulation of its growth in the presence of mercury. A molecular approach to investigate the cadmium tolerance mechanisms was carried out by the application of the suppression subtractive hybridisation (SSH) technique. To this end, we have used a particular strategy to discriminate cadmium-induced differentially expressed genes from those generally expressed upon heavy metal treatment. Several genes (109) were found to be overexpressed in the presence of cadmium, confirming the dramatic metabolic modification underlying the metal stress response of the fungus.

Expression analysis in response to low temperature stress in blood oranges: implication of the flavonoid biosynthetic pathway.

The productivity and the geographical distribution of most commercially important Citrus varieties are markedly affected by environmental low temperatures. As gene engineering has been shown to be a favourable alternative to produce germplasm with improved cold tolerance, a broad group of cold regulated genes have been previously identified from several Citrus spp. By contrast, little information regarding the cold stress response of pigmented sweet orange varieties is available although they might provide a pivotal contribution to define the whole events occurring in cold exposed Citrus fruits. In our work, the transcriptome analysis based on subtractive hybridisation was performed in order to emphasise the overall induction in gene expression after the exposure of blood oranges [(Citrus sinensis) L. Osbeck Tarocco Sciara] to low temperature. The cold induction of several gene expressions was then validated by real-time RT-PCR. Overall, we observed the enhancement of transcripts involved in the defence mechanisms against oxidative damage, osmoregulating processes, lipid desaturation as well as many ESTs implicated in the primary and secondary metabolisms. In particular, the results show that cold stress induces transcriptomic modifications directed towards the increase of flavonoid biosynthesis, including those reactions involved in anthocyanin biosynthesis, as well as of the metabolic pathways supplying it. By comparing the blood orange response to cold stress with those of other plant sources, such as grapefruit, it seems to be similar to that of the chilling acclimated species. Interestingly, among the genes encoding for the regulatory proteins, several transcription factors have been identified for the first time as cold responsive genes in plants, indicating novel investigation lanes which should receive special attention in the future.