Improving sustainable hydrogen production from green waste: [FeFe]-hydrogenases quantitative gene expression RT-qPCR analysis in presence of autochthonous consortia.

BACKGROUND: Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial consortium as an already selected source of implementation for biomass degradation and hydrogen production. The biocatalysts investigated as mainly relevant for hydrogen production were the [FeFe]-hydrogenases expressed in Clostridia, given their very high turnover rates. RESULTS: Bio-hydrogen accumulation was related to the modulation of gene expression of multiple [FeFe]-hydrogenases from two strains (Clostridium beijerinckii AM2 and Clostridium tyrobutyricum AM6) isolated from the same waste. Reverse Transcriptase quantitative PCR (RT-qPCR) was applied over a period of 288 h and the RT-qPCR results showed that C. beijerinckii AM2 prevailed over C. tyrobutyricum AM6 and a high expression modulation of the 6 different [FeFe]-hydrogenase genes of C. beijerinckii in the first 23 h was observed, sustaining cumulative hydrogen production of 0.6 to 1.2 ml H2/g VS (volatile solids). These results are promising in terms of hydrogen yields, given that no pre-treatment was applied, and suggested a complex cellular regulation, linking the performance of dark fermentation with key functional genes involved in bio-H2 production in presence of the autochthonous consortium, with different roles, time, and mode of expression of the involved hydrogenases. CONCLUSIONS: An applicative outcome of the hydrogenases genes quantitative expression analysis can be foreseen in optimising (on the basis of the acquired functional data) hydrogen production from a nutrient-poor green waste and/or low added value compost, in a perspective of circular bioeconomy.

Ligand stabilization and effect on unfolding by polymorphism in human flavin-containing monooxygenase 3.

Pharmacogenomics is a powerful tool to prevent adverse reactions caused by different response of individuals to drug administration. Single nucleotide polymorphisms (SNPs) represent up to 90% of genetic variations among individuals. Drug metabolizing enzymes are highly polymorphic therefore the kinetic parameters of their catalytic reactions can be significantly influenced. This work reports on the unfolding process of a phase I drug metabolizing enzyme, human flavin-containing monooxygenase 3 (hFMO3) and its single nucleotide polymorphic variants (SNPs) V257M, E158K and E308G. Differential scanning calorimetry (DSC) indicates that the thermal denaturation of the enzyme is irreversible. The melting temperature (Tm) for the (Wild Type) WT and its polymorphic variants is found to be in a range from 46 °C to 50 °C. Also the activation energies of unfolding (Ea) show no significant differences among all proteins investigated (290-328 KJ/mol), except for the E308G variant that showed a significantly higher Ea of 412 KJ/mol. The presence of the bound NADP+ cofactor is found to stabilize all the variants by shifting the main Tm by 4-5 °C for all the proteins, exception made for E308G where no changes are observed. Isothermal titration calorimetry (ITC) was used to characterize the interaction of the protein with NADP+ in terms of dissociation constant (Kd), enthalpy (ΔH) and entropy (ΔS). Kd values of 1.6 and 0.7 µM, ΔH of -13.9 Kcal/mol and -16.8 Kcal/mol, ΔS of -20.5 cal/mol/deg, and -28.5 cal/mol/deg were found for V257M and E158K respectively. E308G was found to be unable to bind the NADP+ cofactor, a result that is in line with the Tm results. Circular dichroism also confirmed an overall lower stability of E308G, while NADP+ was found to give a strong positive shift of the Tm stabilizing the structure of E158K (46.2 to 50.6 °C). Previous data highlighted significant differences in terms of activity among the SNPs of hFMO3. In this work a minor impact of the SNPs was found on the stability of the enzyme in the ligand free form, except for E308G, whereas the binding of NADP+ reveals major differences among WT and polymorphic variants that are all measurable in terms of heat capacity, enthalpy and secondary structure content. These data provide the first direct evidence of ligand stabilization effects on hFMO3 that can explain the differences observed in catalytic efficiencies and serve as the starting point for the development of inhibitors of this enzyme.

Influence of different biological control agents and compost on total and nitrification-driven microbial communities at rhizosphere and soil level in a lettuce - Fusarium oxysporum f. sp. lactucae pathosystem.

AIMS: The response of rhizosphere and bulk soil indigenous microbial communities focusing on nitrifiers was evaluated after the application of different biological control agents (BCAs; Bacillus, Trichoderma, Pseudomonas) and compost in controlling lettuce Fusarium wilt. METHODS AND RESULTS: Experiments were conducted 'in situ' over two lettuce cropping seasons. Total fungal, bacterial and archaeal populations and the nitrifiers were analysed using quantitative polymerase chain reaction method. The pathogen, Fusarium oxysporum forma specialis lactucae (FOL), Bacillus, Trichoderma and Pseudomonas and three antifungal genes (chiA, 2,4-diacetylphloroglucinol - phlD and HCN synthase - hcnAB genes) were also assessed. Quantitative data were corroborated with disease severity (DS), potential nitrification activity and soil chemical parameters. The application of BCAs and compost resulted in the disease reduction by as much as 69%, confirmed by significant negative correlations between Bacillus subtilis, Trichoderma and Pseudomonas sp. abundances and DS. The FOL presence in the untreated control resulted in the nitrifiers niche differentiation. CONCLUSIONS: The used treatments were efficient against Fusarium wilt and did not influence negatively the nontarget microbial communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of BCAs and compost appears as an effective and safe strategy to implement sustainable agricultural practices.

Extended C-band tunable multi-channel InP-based coherent receiver PICs.

Fully integrated monolithic, multi-channel InP-based coherent receiver PICs and transceiver modules with extended C-band tunability are described. These PICs operate at 33 and 44 Gbaud per channel under dual polarization (DP) 16-QAM modulation. Fourteen-channel monolithic InP receiver PICs show integration and data rate scaling capability to operate at 44 Gbaud under DP 16-QAM modulation for combined 4.9 Tb/s total capacity. Six channel simultaneous operation of a commercial transceiver module at 33 Gbaud is demonstrated for a variety of modulation formats including DP 16-QAM for >1.2Tbit/s aggregate data capacity.

Root Rot of Spinach in Southern Italy Caused by Pythium aphanidermatum.

During the spring of 2014, spinach (Spinacia oleracea L.) plants of the cv. Crocodile (Rijk Zwaan, De Lier, The Netherlands), grown in a clay loam soil under commercial greenhouse conditions near Salerno (southern Italy), showed stunting, extensive chlorosis, and root rot. Plants were irrigated by overhead sprinklers using well water. Symptoms first developed 20 days after sowing, at air temperatures of 23 to 30°C, and 35% of plants (approximately 15 million plants in 10 ha) were affected. Roots were severely affected, appeared water-soaked and brown, and were characterized by a soft rot. Eventually, affected plants wilted and collapsed. Fifty fragments, each 1 mm2, were excised from symptomatic roots of 10 plants, dipped in a solution containing 1% sodium hypochlorite, rinsed in sterilized water, dried on sterilized paper towel, and plated on both potato dextrose agar (PDA) and the medium BNPRA, which is semi-selective for oomycetes (3). After 5 days of incubation under constant fluorescent light at 22 ± 1°C, 80% of the root sections developed oomycete colonies. One representative isolate, grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup; 15 g agar; 0.5 g CaCO3; 1 liter distilled water) and observed with a light microscope, showed aseptate hyphae 3.3 to 6.5 (mean 5.5) µm wide. Oogonia were globose, smooth, and 22.2 to 31.0 (average 26.3) µm in diameter. Antheridia were barrel-shaped, while oospores were globose and 17.3 to 22.6 (mean 20.9) µm in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of this isolate was amplified using ITS1/ITS4 primers and sequenced. BLAST analysis (1) of the 647-bp segment showed 100% homology with ITS sequences of Pythium aphanidermatum in GenBank (Accession Nos. KJ755088.1, KJ162355.1, KF840479, and KF561235.1). The nucleotide sequence for the Italian spinach isolate was assigned No. KM111256. Pathogenicity tests were performed twice on 20-day-old spinach plants of the cv. Merlo (L'Ortolano, Cesena, Italy), grown in 2-liter pots in a steam-disinfested organic peat substrate (black peat, pH 6.5 to 6.8, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) moistened to field capacity, and infested with wheat and hemp kernels colonized with isolate Py 1-14 of P. aphanidermatum at 1 g/liter. Five plants were transplanted into each of four pots filled with infested peat, while the same number of plants was grown in non-infested substrate as a control. Plants were kept in two growth chambers, each with 12 h of light per day at 20 or 30°C, and were irrigated daily to maintain the potting medium at field capacity. Symptoms first developed 5 days after the spinach was transplanted into infested potting medium in the growth chamber maintained at 30°C. After 10 days, all plants in this growth chamber were dead, while only 5% of the plants growing in infested potting medium in the 20°C growth chamber were affected. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in infested medium. No fungi were re-isolated from the asymptomatic control plants grown in non-infested substrate. To our knowledge, this is the first report of P. aphanidermatum causing root rot on S. oleracea in Italy. The same pathogen has been reported to cause root rot of spinach in other countries, including the United States (2). The disease is, at present, limited to the affected greenhouses observed in this study. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) M. L. Bates and M. E. Stanghellini. Plant Dis. 68:989, 1984. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, FL., 2002.

PRE-PLANTING TREATMENTS WITH PHOSPHITE-BASED PRODUCTS AGAINST DIFFERENT FOLIAR AND SOIL-BORNE PATHOGENS OF VEGETABLE CROPS.

Fifteen experimental trials were carried out under greenhouse conditions to evaluate the efficacy of preventative treatments based on phosphite salts on the following pathosystems: tomato/Phytophthora nicotianae, zucchini/P. capsici, lettuce/Fusarium oxysporum f.sp. Iactucae, rocket/Fusarium oxysporum f. sp. raphani, wild rocket/Plectosphaerella cucumerina and basii/Peronospora belbahrii. The possible use of phosphite salts in nursery cultivation systems is considered in comparison with chemical fungicides. Phosphites-based products reduced 66-88% and 56-72% the severity of Phytophthora crown root rot of tomato and zucchini, respectively. Four application with the phosphites-based products provided a disease reduction of Fusarium wilt of lettuce from of 33 to 83% and of 45 to 68% on cultivated rocket. These products provide the most constant results when applied in three treatments against Plectosphaerella cucumerina with a disease reduction ranging between 34%-82%. Phosphite-based products showed results statistically similar to mefenoxam when tested against downy mildew of basil. Their contribution to disease management can be very interesting, because they can complement other control measures.

EFFECT OF BIOSOLARISATION ON THE MICROBIAL POPULATIONS OF SUBSTRATES INFESTED WITH FUSARIUM OXYSPORUM BY PCR-DGGE.

Biosolarisation consists of combining solarisation and organic matter application for controlling soilborne pathogens. The effects of this control strategy on the microbial community is almost unknown and needs to be investigated with molecular tools. The aim of the research was to investigate how biosolarisation can affect the structure of the microbial populations evaluated by a culture independent method using DGGE of PCR-amplified 18S-ITS genes-coding fragments from DNA extracted directly from infested substrate. Substrate samples were artificially infested with Fusarium oxysporum f. sp. conglutinans (FOC) and F. oxysporum f.sp. basilici (FOB) in order to evaluate the shift in fungal population by using culture independent methods. Solarisation was carried out with transparent polyethylene film during the summer period in a greenhouse located in Northern Italy, in combination or not with Brassica carinata defatted seed meals and/or compost. Biosolarisation treatment was carried out in a growth chamber by heating the substrate for 7 and 14 days at optimal (55-52 degrees C for 6 h, 50-48 degrees C for 8 h and 47-45 degrees C for 10 h/day) and sub-optimal (50-48 degrees C for 20 h, 45-43 degrees C for 8 h and 40-38 degrees C for 10 h/day) temperatures. Plate counts and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analyses were performed to evaluate the effect of biosolarisation on the microbial population. The abundance of FOC and FOB was reduced as a consequence of biosolarisation, while bacterial populations were higher compared to control samples during the experiment. PCR-DGGE fingerprints of the ascomycete community obtained from DNA directly extracted from infested substrate samples showed that the use of organic amendments increased the similarity of the fungal populations.

First Report of Root Rot Caused by Pythium aphanidermatum on Bell Pepper (Capsicum annuum L.) in Italy.

During July 2012, symptoms of root rot were observed on bell pepper (Capsicum annuum) grown in 2,000 m2 of commercial greenhouses near Cuneo in northern Italy. Symptoms first developed 30 to 40 days after transplanting, when greenhouse temperatures ranged from 25 to 30°C, and 10% of the plants were affected. Affected plants were stunted with leaf chlorosis, reduced growth, and sudden wilting. Roots were severely affected with a brown discoloration, water-soaking, and soft rot. Eventually, affected plants collapsed. Tissue fragments of 1 mm2 were excised from symptomatic roots, dipped in a 1% sodium hypochlorite solution, and placed on potato dextrose agar (PDA) and an agar medium selective for oomycetes (3). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. An isolate grown for 12 days on V8 agar medium (200 ml V8 Campbell Soup, 15 g agar, 0.5 g CaCO3, and 1 liter distilled water) showed aseptate hyphae that were 3.5 to 6.3 µm (avg. 5.2 µm) wide. Oogonia were globose, smooth, and 24.3 to 29.0 (avg. 25.1) µm in diameter. Antheridia were barrel-shaped, while oospores were globose, and 17.3 to 23.5 µm (avg. 21.2 µm) in diameter. These morphological characters identified the microorganism as a Pythium sp. (4). The ITS region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 781-bp segment (GenBank Accession KF840479) showed 100% homology with the ITS sequence of an isolate of Pythium aphanidermatum in GenBank (AY598622.2). Pathogenicity tests were performed twice on 30-day-old plants of C. annuum cv. Cuneo grown in 2-L pots (4 plants/pot), containing a steam-disinfested, organic peat substrate (70% black peat and 30% white peat, pH 5.5 to 6.0, N 110 to 190 mg/liter, P2O5 140 to 230 mg/liter, K2O 170 to 280 mg/liter) that was infested with wheat and hemp kernels colonized by the isolate of P. aphanidermatum, at a rate of 1 g colonized kernels/liter potting medium. The inoculum was prepared by autoclaving at 121°C for 30 min a mixture of wheat-hemp kernels (2:1 v/v) in a 1-liter flask, to which the bell pepper isolate of P. aphanidermatum was added in the form of colonized agar medium selective for oomycetes plugs. Before use, the inoculated flask was incubated for 10 days at 22°C in the dark. Four plants/pot were transplanted into each of four pots filled with the infested medium/growth chamber, while the same number of plants were grown in non-infested substrate in pots in each growth chamber. Plants were kept in two growth chambers, one set at 20°C and the other at 28°C. Symptoms first developed 7 days after inoculation. After 30 days, 50% of inoculated plants showed brown roots and died in the growth chamber set at 28°C, while only 10% of the plants were symptomatic at 20°C. Control plants remained asymptomatic at both temperatures. P. aphanidermatum was re-isolated consistently from the symptomatic roots of plants grown in the infested soil by using the same protocol as the original isolations, while no fungal colonies were obtained from asymptomatic roots of the non-inoculated control plants. To our knowledge, this is the first report of the presence of P. aphanidermatum on C. annuum in Italy. The same disease was reported in the United States (2). The importance of the disease, although limited in distribution at present to the greenhouses surveyed in northern Italy, could increase in areas where sweet pepper is grown intensively. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. O. Chellemi et al. Plant Dis. 84:1271, 2000. (3) H. Masago et al. Phytopathology 67:425, 1977. (4) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Boca Raton, FL, 2002.

VARIETAL RESISTANCE TO CONTROL FUSARIUM WILTS OF LEAFY VEGETABLES UNDER GREENHOUSE.

Fusarium wilts of leafy vegetables are difficult to manage under intensive cropping systems. The objective of this study was to evaluate, in three experimental trials, the susceptibility of commercial cultivars of lettuce, wild and cultivated rocket and lamb's lettuce to Fusarium wilts in orderto provide information to breeders, as well as to growers. Some of the cultivars of lettuce tested were completely resistant to the three races of Fusarium wilt. It is interesting to observe that most of the resistant cultivars were 'Batavia red'. Only few rocket cultivars commercially available show a partial resistant reaction to F. oxysporum f.sp. raphani, while, varietal resistance is not applicable at the moment to control Fusarium wilt of lamb's lettuce. The integration of cultural practices, use of resistant cultivars, when available, chemicals and biological control agents, permit to prevent and manage these important diseases on leafy vegetables for fresh-cut production.

CONTROL OF SOIL-BORNE DISEASES BY DIFFERENT COMPOSTS IN POTTED VEGETABLE CROPS.

The composting process and the type and nature of wastes and raw materials influence the maturity, quality and suppressiveness of composts. Variability in disease suppression also depends on the pathosystem, on soil or substrate type, on chemical-physical conditions, like pH and moisture, and on the microbial component of compost. The aim of the research was to evaluate the suppressiveness of composts, originated from green wastes and/or municipal biowastes, and produced by different composting plants located in Europe. The composts were tested against soil-borne pathogens in greenhouse on potted plants: Fusarium oxysporum f.sp. busilici/basil, Pythium ultimum/cucumber, Rhizoctonia solani/bean. Composts were blended with a peat substrate at different dosages (10, 20 and 50% vol./vol.) 14 days before seeding or transplanting. Pythium ultimum and Rhizoctonia solani were mixed into the substrate at 0.5 g of wheat kernels L(-1) 7 days before seeding, while, in the case of Fusarium oxysporum f.sp. basilici, chlamydospores were applied at 1 x 10(4) CFU/g. Seeds of basil, cucumber and bean were sown into 2 L pots in greenhouse. The number of alive plants was counted and above ground biomass was weighed 30 days after seeding. The number of infected cucumber and basil plants was significantly reduced by increasing dosages of composts, but municipal compost was phytotoxic when applied at high dosages compared to green compost. Moreover, municipal compost increased the disease caused by Rhizoctonia solani on bean. The use of compost in substrates can be a suitable strategy for controlling soil-borne diseases on vegetable crops, but results depend on type of composts, application rates and pathosystems.

Effect of silicates and electrical conductivity on Fusarium wilt of hydroponically grown lettuce.

Silicon can stimulate natural defense mechanisms in plants, reducing foliar diseases like powdery arid downy mildew on several crops, including lettuce. The effect of silicate on Fusarium wilt, caused by Fusarium oxysporum f. sp. lactucae was evaluated under greenhouse conditions on lettuce grown in soilless systems. Silicon, as potassium silicate, was added at 100 mg L(-1) of nutrient solution at three levels of electrical conductivity; 1.5-1.6 mS cm(-1) (E.C.1), 3.0-3.2 mS cm(-1) (E.C.2) and 4-4.2 mS cm(-1) (E.C.3). Pots containing lettuce plants were first inoculated with F. oxysporum f. sp. lactucae (3x10(5) chlamidospores ml(-1)) 15-20 days before transplanting. Disease severity and physiological parameters, including chlorophyll content, were analyzed weekly after transplanting. The addition of potassium silicate slightly reduced Fusarium wilt, at all levels of electrical conductivity under study, compared to the control. On the contrary, the increase of electrical conductivity of the nutrient solution showed no effect on the disease. The use of silicon was previously demonstrated to significantly reduce downy mildew on lettuce in soilless systems, and in this trial it demonstrated to slightly reduce disease severity of an important soil-borne pathogen like F. oxysporum f. sp. lactucae, suggesting the possibility to apply it successfully in soilless crops.

First Report of Southern Blight Caused by Sclerotium rolfsii on Common Bean (Phaseolus vulgaris) in Italy.

Common bean (Phaseolus vulgaris L.) is grown worldwide for consumption of dry or green beans. During late spring of 2012, yellowing and wilting symptoms were observed in a commercial bean field cv. Lingua di fuoco in Cagliari Province (Sardinia, southern Italy) on 30% of plants 4 to 5 months after sowing. The first symptoms developed in May, when temperatures reached 18 to 30°C. Affected plants showed crown rot, necrosis of the cortex, and foliar chlorosis. As disease progressed, plants collapsed. In the presence of abundant moisture, white mycelium developed on the senescent tissue along with light to dark brown sclerotia (3.0 to 4.8 mm in diameter). Symptomatic tissue was disinfested for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 25 mg streptomycin sulfate/liter. The fungus that was isolated consistently from symptomatic plants onto PDA at 23°C grew rapidly in culture with silky-white, sterile mycelium, formed light to dark brown sclerotia (each 1.8 to 3.2 mm in diameter) after 7 days, and readily produced aerial hyphae. These morphological features are typical of Sclerotium rolfsii (2). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified for one isolate using ITS1/ITS4 primers (4), and sequenced (GenBank Accession No. KF002510). BLASTn analysis (1) of the 656-bp segment showed 87% homology with the ITS sequence of an S. rolfsii isolate (JF819727). Pathogenicity of one isolate was confirmed by inoculating healthy P. vulgaris plants cv. Lingua di fuoco grown in 2-liter pots in a steamed potting mix containing 50% Tecno2 (70% white peat and 30% clay) and 50% Tiesse 3 (60% white peat, 20% clay, and 20% perlite) (Turco Silvestro terricci, Bastia d'Albenga, SV, Italy). Inoculum consisting of mycelium and sclerotia of the pathogen produced from 10-day-old cultures on PDA was mixed in the soil at 0.5 g/liter substrate. Four 7-day-old plants per pot, with three replicate pots, were used for inoculation. The same number of control plants grown in the same substrate were inoculated with non-colonized PDA as a negative control treatment. The pathogenicity test was repeated. Plants were kept in a growth chamber at 30°C and 85% RH. Inoculated plants developed symptoms of leaf yellowing within 10 days, followed by crown rot, appearance of white mycelium and sclerotia, and eventual wilting. Control plants remained asymptomatic. Isolations from inoculated plants demonstrated the absence of latent infections by the fungus S. rolfsii, but the fungus was not reisolated from non-inoculated control plants. To our knowledge, this is the first report of S. rolfsii infecting P. vulgaris in Italy. Southern blight has been reported on common bean in sub-tropical and tropical areas of the world (3), where it can cause severe crop losses. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) J. E. M. Mordue. CMI Descriptions of Pathogenic Fungi and Bacteria No. 410, 1974. (3) H. F. Schwartz et al. Page 20 in: Compendium of Bean Diseases. American Phytopathological Society Press, St. Paul, MN, 2005. (4) T. J. White et al. PCR Protocols. Page 315 in: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Damping-off Caused by Pythium aphanidermatum on Leaf Beet (Beta vulgaris subsp. vulgaris) in Italy.

During July 2010, symptoms of crown and root rot were observed on leaf beet (Beta vulgaris L. subsp. vulgaris) grown in a commercial field near Torino (northern Italy). The first symptoms developed 25 days after sowing with temperatures ranging from 25 to 30°C, and 20% of plants were affected. Affected plants were stunted and leaves showed chlorosis and suddenly wilted. The collar and young stems were affected first and appeared brown, water-soaked, and were characterized by a soft rot. Eventually, all affected plants collapsed. Thin aerial mycelia were visible on the surface of the infected plants if maintained at a high relative humidity. Tissue fragments of 1 mm2 were excised from the margins of the lesions, dipped in a solution containing 1% sodium hypochlorite, and plated on potato dextrose agar (PDA) and on a medium selective for oomycetes (2). Plates were incubated under constant fluorescent light at 22 ± 1°C for 5 days. Five isolates, grown on V8 medium (vegetable mix 300 g; agar 15 g; CaCO3 1.5 g; distilled water 1 liter) and observed under light microscope showed the morphological characters of Pythium aphanidermatum (3). This result was confirmed by PCR and sequence analysis. The internal transcribed spacer (ITS) region of rDNA of a single isolate (Py 7/10) was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 815 bp segment showed a 99% homology with the sequence of P. aphanidermatum (GenBank Accession JN695786). The nucleotide sequence has been assigned to the GenBank Accession JX462954. Pathogenicity tests were performed twice on B. vulgaris subsp. vulgaris grown in 2-liter pots, containing a steam disinfested organic peat substrate (70% black peat, 30% white peat, pH 5.5 to 6, N 110 to 190 mg L-1, P2O5 140 to 230 mg L-1, K2O 170 to 280 mg L-1), infested with wheat and hemp kernels colonized with a strain of P. aphanidermatum at a rate of 1 g L-1. Ten seeds per pot were sown in four pots filled with the infested medium, while the same number of seeds were sown in non-infested substrate. Plants were kept in two growth chambers, at 20 and 27°C. The first symptoms developed 7 days after the artificial inoculation. After 20 days, 70% of plants were infected at 27°C, while 10% were affected at 20°C. Control plants remained healthy at both temperatures. P. aphanidermatum was consistently reisolated from the lesions. To our knowledge, this is the first report of damping off of B. vulgaris subsp. vulgaris caused by P. aphanidermatum in Italy. The importance of the disease, at present limited, could increase in areas where leaf beet is intensively grown. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. Masago et al. Phytopathology 67:425, 1977. (3) T. Watanabe. Pictorial Atlas of Soil and Seed Fungi. CRC Press, Florida, 2002.

First Report of a New Leaf Spot Caused by Plectosphaerella cucumerina on Field Grown Endive (Cichorium endivia) in Italy.

During summer 2012, symptoms of a new leaf spot disease were observed in several commercial fields in Treviglio (Bergamo, northern Italy) on plants of curly (Cichorium endivia var. crispum) and Bavarian (C. endivia var. latifolium) endive (Asteraceae). This crop is widely grown in the region for fresh market. The first symptoms on leaves of affected plants consisted of small (1 mm) black-brown spots of irregular shape, later coalescing into larger spots, up to 10 to 15 mm diameter. Eventually, spots were surrounded by a yellow halo. Particularly, affected tissues rotted quickly under high moisture. Disease severity was greatest at 75 to 90% RH and air temperature between 23 and 30°C, where affected tissues rotted quickly. This disease resulted in severe production losses. On one farm in particular, three different fields totaling 2 ha, 5 to 13% of the plants were affected. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then placed on potato dextrose agar (PDA) medium, containing 25 mg/liter of streptomycin sulphate. After 5 days, a fungus developed producing a whitish-orange mycelium when incubated under 12 h/day of fluorescent light at 23°C. The isolates obtained were purified on PDA. On this medium, they produced hyaline elliptical and ovoid conidia, rarely septate, measuring 5.0 to 9.0 × 1.7 to 3.9 (average 6.0 × 2.9) µm. Conidia were born on phialides, single, clavate, and 2.8 × 1.4 µm. Such characteristics are typical of Plectosphaerella sp. (1,2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (3) and sequenced. BLAST analysis of the 530-bp segment obtained from C. endivia var. crispum isolate PLC28 and of the 527-bp from C. endivia var. latifolium isolate PLC 30, respectively, showed 99% similarity with the sequence of Plectosphaerella cucumerina (anamorph Plectosporium tabacinum), GenBank EU5945566. The nucleotide sequences of isolates PLC 28 and PLC 30 have been assigned the GenBank accession numbers KC293994 and KC293993, respectively. To confirm pathogenicity, tests were conducted on 30-day-old C. endivia plants. C. endivia var. crispum cv Myrna and C. endivia var. latifolium cv. Sardana plants, grown in 2-liter pots (1 plant per pot, 10 plants per treatment) were inoculated by spraying a 106 CFU/ml conidial suspension of the two isolates of P. cucumerina, prepared from 10-day-old cultures, grown on PDA. Inoculated plants were maintained in a growth chamber at 25 ± 1°C and 90% RH for 5 days. Non-inoculated plants, only sprayed with water, served as controls. All plants inoculated with the two isolates, showed typical leaf spots 7 days after the artificial inoculation, similar to those observed in the field. Later, spots enlarged and leaves rotted. Non-inoculated plants remained healthy. P. cucumerina was reisolated from inoculated plants. The pathogenicity tests were conducted twice with identical results. This is, to our knowledge, the first report of P. cucumerina on endive n Italy, as well as worldwide. Due to the importance of the crop in Italy, this disease can cause serious economic losses. References: (1) A. Carlucci et al. Persoonia 28:34, 2012. (2) M. E. Palm et al. Mycologia 87:397, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Leaf Spot of Lettuce (Lactuca sativa L.) Caused by Phoma tropica in Italy.

Lettuce (Lactuca sativa L.) is widely grown in Italy, with the production for the preparation of ready-to-eat salads becoming increasingly important. During the spring of 2011, a previously unknown leaf spot was observed on L. sativa plants, cv Rubia, grown in several plastic tunnels in Lumbardy (northern Italy), 20 to 25 days after sowing. Thirty to forty per cent of leaves of the plants growing in the part of the tunnel with the highest relative humidity were affected. Leaves of infected plants showed extensive, irregular, dark brown, necrotic lesions with a chlorotic halo. Lesions initially ranged from 0.5 to 3 mm, then eventually coalesced, reaching 2 to 3 cm, showing a well-defined, dark brown border. Affected leaves senesced and withered. The crown was not affected by the disease. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then cultured on potato dextrose agar (PDA), amended with 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed, producing a greenish grey mycelium with a white border when incubated under 12 h/day of fluorescent light at 21 to 23°C. In order to favor the production of conidia, the fungus was transferred on malt extract agar (MA) and maintained under 12 h/day of fluorescent light at 22°C. After 15 days, black pycnidia, 175 to 225 µm, developed, with hyaline, elliptical, unicellular conidia, measuring 3.21 to 6.7 × 1.08 to 3.2 (average 5.5 × 1.9) µm. On the basis of these morphological characteristics, the fungal causal agent of the disease could be related to the genus Phoma (2). The internal transcribed spacer (ITS) region of rDNA of the isolate PHT30 was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 466-bp segment showed a 99% similarity with the sequence of Phoma tropica (GenBank Accession No. JF923820.1). The nucleotide sequence has been assigned the GenBank Accession No. JQ954396. Pathogenicity tests were performed by spraying healthy 20-day-old lettuce plants, cv Rubia, with a spore suspension (1 × 105 conidia/ml) prepared from 14-day-old colonies of the strain PHT30 grown on MA cultures. Plants inoculated with water alone served as controls. Ten plants per isolate were used. Plants were covered with plastic bags for 5 days after inoculation and maintained in a growth chamber at 20°C and 80% relative humidity. The first foliar lesions, similar to those occurring on the naturally infected plants, developed on leaves 12 days after inoculation. Control plants remained healthy. The pathogen was consistently reisolated from leaf lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of P. tropica on lettuce in Italy as well as worldwide. In the United States, the presence of P. exigua was reported in 2006 (3). The economic importance of the disease at present is limited, probably also because symptoms can be confused with those caused by Botrytis cinerea. However, P. tropica could become a more significant problem because of the importance of the crop. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) G. H. Boerema. Trans. Br. Mycol. Soc. 67:289, 1976. (3) S. Y. Koike. Plant Dis. 90:1268, 2006.

Phytophthora capsici: A Soilborne Pathogen Dangerous on Grafted Tomato (Solanum lycopersicum × S. hirsutum) in Italy.

During an extensive survey carried out in Piedmont (northern Italy) aimed at identifying the emerging soilborne diseases affecting tomato in commercial fields where alternatives to methyl bromide have been implemented in response to national and international regulations, sudden collapse of tomato plants, cv. Tomahawk, grafted on cv. Beaufort, were repeatedly observed in a commercial plastic tunnel operation. Affected plants suddenly collapsed 60 days after transplant during the month of May 2010. Symptoms included chlorosis, stunting, and severe root and crown rot, leading to sudden collapse of approximately 25% of the plants within 60 days of transplant. Symptomatic tissues from the root and collar of infected plants were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in water, and submerged in selective medium based on corn meal agar. A Phytophthora-like organism (2) with characteristic coenocytic hyphae was consistently isolated and transferred to V8 agar. The sporangia were spherical to ovoid, papillate, and 40 to 77 × 23 to 34 (average 55.1 × 30.3) µm. Oospores were globose and 22.2 to 30.8 µm. The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 750-bp segment showed a 100% homology with the sequence of Phytophthora capsici JN382543.1. The nucleotide sequence has been assigned the GenBank Accession No. JX090306. Pathogenicity tests were performed on healthy 30-day-old tomato plants cv. Beaufort by using one strain of P. capsici grown for 15 days at 22 to 25°C on a mixture of 2:1 wheat/hemp kernels, and then 1 g per L of the inoculum was mixed into a substrate based on peat blonde/peat black (15:85 v/v). Two plants were transplanted into 3-L pots, with five replicates. Ten non-inoculated plants represented the control treatment; the trial was repeated once. All plants were kept in a greenhouse at temperatures ranging from 22 to 25°C. Inoculated plants became chlorotic 7 days after inoculation and root and crown rot developed 30 days after inoculation. Control plants remained symptomless. P. capsici consistently was reisolated from inoculated plants. In Italy, the presence of P. nicotianae on hybrids of Solanum lycopersicum × S. hirsutum is known (3), while, to the best of our knowledge, this is the first report of P. capsici on the hybrid S. lycopersicum × S. hirsutum in Italy. The economic importance of the disease can increase due to the expanding use of grafted tomato plants. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St Paul, MN, 1996. (3) A. Garibaldi and M. L. Gullino. Acta Hortic. 833:35, 2010. (4) H. M. Masago et al. Phytopathology 67:425, 1977.

First Report of Plectosphaerella cucumerina on Greenhouse Cultured Wild Rocket (Diplotaxis tenuifolia) in Italy.

During spring 2012, symptoms of an unusual leaf spot disease were observed in several commercial greenhouses near Salerno (southern Italy) on plants of Diplotaxis tenuifolia (cv Selvatica). The first symptoms on leaves of affected plants consisted of small (1 mm) black-brown spots of irregular shape, later coalescing into larger spots, 1 cm in diameter. Spots were surrounded by a yellow halo, and were mostly located on the foliar limb, rib, and petiole. Affected leaves were often distorted, appearing hook-like. The disease was severe under 75 to 90% RH, at air temperature of 20 to 26°C, and caused severe production losses on about 50 ha. Particularly, affected tissues rotted quickly after packaging and during transit and commercialization of processed rocket. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then placed on potato dextrose agar (PDA) medium, containing 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed producing a whitish-orange mycelium when incubated under 12 h/day of fluorescent light at 22°C. The isolates obtained were purified on PDA. On this medium, they produced hyaline elliptical and ovoid conidia, sometimes one-septate, measuring 4.5 to 9.2 × 1.7 to 3.5 (average 6.8 × 2.6) µm. Conidia were born on phialides, measuring 6.8 to 20.2 × 1.3 to 3.1 (average 16.5 × 2.1) µm. Such characteristics are typical of Plectosphaerella sp. (2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 (3) and sequenced. BLAST analysis of the 519-bp segment showed a 98% similarity with the sequence of Plectosphaerella cucumerina (GenBank Accession No. AB469880). The nucleotide sequence has been assigned the GenBank Accession JX185769. To confirm pathogenicity, tests were conducted on 45-day-old D. tenuifolia plants. Plants (21/treatment), grown in 15 liter pots (7 plants/pot) were inoculated by spraying a 1 × 106 CFU/ml conidial suspension of one isolate of P. cucumerina, prepared from 10-day-old cultures, grown on PDA. Inoculated plants were maintained in a growth chamber at 23 ± 1°C, at 90% RH for 4 days. Non-inoculated plants served as control. Inoculated plants showed the typical first leaf spots 6 days after the artificial inoculation. Four days later, spots enlarged and leaves became distorted, showing chlorosis. Non-inoculated plants remained healthy. P. cucumerina was reisolated from inoculated plants. The pathogenicity test was conducted twice with identical results. This is, to our knowledge, the first report of P. cucumerina on D. tenuifolia in Italy as well as worldwide. P. cucumerina has been described as associated with root and collar rots of other horticultural crops in southern Italy (1). Due to the importance of the crop in Italy, this disease can cause serious economic losses. References: (1) A. Carlucci et al., Persoonia, 28:34, 2012. (2) M. E. Palm et al. Mycologia, 87:397, 1995. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Rust Caused by Pucciniastrum circaeae on Fuchsia × hybrida in Italy.

Fuchsia is a genus of flowering plants that is native to South America and New Zealand and belongs to the family Onagraceae. In September 2011, 2-year-old potted plants of Fuchsia × hybrida, cv. Citation, in a garden located near Biella (northern Italy) showed signs and symptoms of a previously unknown disease. Typically, infected plants showed leaf chlorosis followed by the appearance of necrosis on the adaxial leaf surfaces, while the abaxial surfaces showed orange uredinia irregularly distributed. As the disease progressed, infected leaves turned yellow and wilted. Affected plants showed a progressive phylloptosis and also flowering was negatively affected. Urediniospores were globose, yellow to orange, and measured 14.6 to 25.9 (average 19.6) µm. Teliospores were not observed. Morphological characteristics of the fungus corresponded to those of the genus Pucciniastrum. DNA extraction and PCR amplification were carried out with Terra PCR Direct Polymerase Mix (Clontech, Saint Germain-en-Laye, France) and primers ITS1/ITS4 (4). A 700-bp PCR product was sequenced and a BLASTn search (1) confirmed that the sequence corresponded with a 96% identity to Pucciniastrum circaeae. The nucleotide sequence has been assigned the GenBank Accession No. JQ029688. Pathogenicity tests were performed by spraying leaves of healthy 1-year-old potted Fuchsia × hybrida plants with an aqueous suspension of 1 × 103 urediniospores ml-1. The inoculum was obtained from infected leaves. Plants sprayed only with water served as controls. Three plants were used for each treatment. Plants were covered with plastic bags for 4 days after inoculation and maintained outdoors at temperatures ranging between 18 and 25°C. Lesions developed on leaves 20 days after inoculation with the urediniospore suspension, showing the same symptoms as the original plants, whereas control plants remained healthy. The organism that was recovered from the lesions after inoculation was the same as the one obtained from the diseased plants. The pathogenicity test was carried out twice with similar results. The presence of P. fuchsiae, later identified as P. epilobii, was repeatedly reported in the United States (3). P. epilobii and P. circaeae have closely related hosts and morphologically similar urediniospores. These species were reported to form a single group in molecular phylogenetic trees (2). This is, to our knowledge, the first report of P. circaeae on Fuchsia × hybrida in Italy. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) Y. M. Liang et al. Mycoscience 47:137, 2006. (3) L. B. Loring and L. F. Roth. Plant Dis. Rep. 48:99, 1964. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

P450-based porous silicon biosensor for arachidonic acid detection.

A porous silicon biosensor based on P450 enzyme for arachidonic acid detection was developed. A new transduction method is presented with a simultaneous measurement of refractive index and fluorescence intensity changes when the analyte is binding to an enzyme on the porous silicon surface. A fluorophore bound to a cysteine residue in an allosteric position of the haem domain (BMP) of cytochrome P450 BM3 enhances its fluorescence intensity upon interaction with its substrate arachidonic acid, involved in diseases such as Alzheimer's, liver cancer and cellular inflammation processes. BMP has been anchored on porous silicon surface and the new transduction method has been successfully exploited to develop a biosensor for arachidonic acid, reaching a detection limit of 10 µM arachidonic acid in a dynamic range of 10-200 µM. Moreover, the change of the refractive index has been also monitored at the same time, displaying a higher detection limit of 30 µM. Preliminary test were also conducted in plasma proving the high specificity and selectivity of the sensor even in presence of interferents in the range of 50-100 µM. Here we suggest these two detection systems could be used simultaneously to increase the accuracy and the dynamic range of the sensor avoiding a false positive response.