On the prediction of non-CG DNA methylation using machine learning.

DNA methylation can be detected and measured using sequencing instruments after sodium bisulfite conversion, but experiments can be expensive for large eukaryotic genomes. Sequencing nonuniformity and mapping biases can leave parts of the genome with low or no coverage, thus hampering the ability of obtaining DNA methylation levels for all cytosines. To address these limitations, several computational methods have been proposed that can predict DNA methylation from the DNA sequence around the cytosine or from the methylation level of nearby cytosines. However, most of these methods are entirely focused on CG methylation in humans and other mammals. In this work, we study, for the first time, the problem of predicting cytosine methylation for CG, CHG and CHH contexts on six plant species, either from the DNA primary sequence around the cytosine or from the methylation levels of neighboring cytosines. In this framework, we also study the cross-species prediction problem and the cross-context prediction problem (within the same species). Finally, we show that providing gene and repeat annotations allows existing classifiers to significantly improve their prediction accuracy. We introduce a new classifier called AMPS (annotation-based methylation prediction from sequence) that takes advantage of genomic annotations to achieve higher accuracy.

N-Source Determines Barley Productivity, Nutrient Accumulation, and Grain Quality in Cyprus Rainfed Agricultural Systems.

The Eastern Mediterranean and Middle East (EMME) region is already experiencing the negative effects of increased temperatures and the increase in prolonged drought periods. The use of organic fertilization could be a valuable tool to meet the main challenges of climate change and maintain the productivity, quality, and sustainability of rainfed agricultural ecosystems. In the current study, we compare the effect of manure, compost, and chemical fertilization (NH4NO3) on barley grain and straw yield in a field study for three consecutive growing seasons. The hypothesis that the barley productivity, nutrient accumulation, and grain quality remain similar among the different nutrient management strategies was tested. The results showed that both growing season and type of nutrient source significantly affected barley grain and straw yield (F6,96 = 13.57, p < 0.01). The lowest productivity was noticed in the non-fertilized plots while chemical and organic fertilization exhibited similar grain yield, ranging from 2 to 3.4 t/ha throughout the growing seasons. For straw, the use of compost had no effect on the yield in any of the growing seasons examined. The use of manure and compost had a significant effect on grain macro- and micronutrient content but this was highly related to growing season. Principal component analysis (PCA) clearly demonstrated the discrimination of the different type of fertilization on barley performance during the course of the study, while the application of compost was highly associated with an increase in micronutrients in grain samples. Furthermore, structural equational modeling (SEM) showed that both chemical and organic fertilization had a direct positive effect on macro- (r = 0.44, p < 0.01) and micronutrient (r = 0.88, p < 0.01) content of barley grain and a positive indirect effect on barley productivity through N accumulation in grain (ß = 0.15, p = 0.007). The current study showed that barley grain and straw yield was similar between manure and NH4NO3 treatments, while compost exhibited a residual positive effect causing an increase in grain yield during the growing season. The results highlight that N fertilization under rainfed conditions is beneficial to barley productivity through its indirect effects on N accumulation in grain and straw, while it improves grain quality through the increased accumulation of micronutrients.

LCA of Barley Production: A Case Study from Cyprus.

Greenhouse gas emissions (i.e., carbon dioxide, methane, nitrous oxide) produced by agriculture contribute to global warming and climate change. Various practices followed by farmers in different environmental conditions contribute to the increase in the phenomena, and there is a need for immediate measures. The current study examines the environmental impact of barley production under rain-fed conditions in Cyprus. For this, four different nutrient management scenarios were investigated in order to evaluate the environmental performance of crop production, namely: (1) Nitrogen (20%), Phosphorous (20%), Potassium (10%); (2) Nitrogen (20%), Phosphorous (20%), Potassium (10%) and manure; (3) Nitrogen (25%), Phosphorous (10%), Potassium (0%); and (4) Nitrogen (25%), Phosphorous (10%), Potassium (0%) and manure. Data were collected from two different areas of Cyprus (Nicosia and Larnaca) through on-site visits and questionnaires. Life Cycle Assessment (LCA) was used as a method to quantify environmental impacts which were categorized into six impact categories: (i) acidification potential (AP), (ii) eutrophication potential (EP), (iii) global warming potential (GWP), (iv) ozone depletion potential (ODP), (v) photochemical, ozone creation potential (POCP), and (vi) terrestrial ecotoxicity (TAETP). LCA was used with system boundaries from field to harvest and a functional unit (FU) of one bale of hay. Research results showed that the addition of manure increased values in all impact categories. Comparing scenarios without manure (1 and 3) and with manure (2 and 4), the main process which contributed to GWP was field preparation, which resulted in 3 t CO2-Eq∙FU-1 and 46.96 t CO2-Eq∙FU-1, respectively. Furthermore, the highest contribution of sub-processes to GWP (kg CO2-Eq∙FU-1) was machinery maintenance (scenarios 2 and 4). The potential to reduce environmental impacts from barley and moreover, to mitigate the footprint of the agriculture sector in Cyprus is proposed by changing existing practices such as decreasing fuel consumption by agricultural machinery, and monitoring fertilizing and seeding. Conclusively, the carbon footprint of barley can be decreased through the improvement of nutrient management and cropping practices.

Differential response of N2O emissions, N2O-producing and N2O-reducing bacteria to varying tetracycline doses in fertilized soil.

Tetracyclines are the most widely used antibiotics worldwide. Their presence in soils could affect nutrient cycling, but our knowledge regarding how they affect soil microbial communities involved in greenhouse gas emissions is limited. The objective of the current study was to evaluate how tetracycline is affecting N2O emissions and the abundance of denitrifiers in fertilized soil. For this purpose, soil mesocosms were treated with only NH4NO3 (100 mg/kg) or NH4NO3 (100 mg/kg) plus three different doses of tetracycline (0.1, 0.5 and 2 mg/kg). Soils that did not receive tetracycline or NH4NO3 were used as controls. Nitrous oxide fluxes were monitored daily for 16 days. The total bacterial (16S rRNA), the abundance of N2O-reducing and -producing bacteria were quantified by qPCR at the end of the experiment. The application of NH4NO3 caused a significant increase of N2O emissions and AOB abundance but did not affect the abundance of denitrifiers and AOA compared to control soils. Different doses of tetracycline in fertilized soils did not mitigate these N2O emissions; instead, higher cumulative emissions were noticed in soils treated with the lowest dose. In these soils the total bacterial abundance was higher compared to soils received higher tetracycline concentration. The abundances of the N2O-producing and N2O-reducing communities were also differently affected by the addition of tetracycline, which was dose-dependent. Higher doses of tetracycline favored N2O-reducers within the total bacterial community, which could be important for mitigating N2O emissions in the long term.

Land use in urban areas impacts the composition of soil bacterial communities involved in nitrogen cycling. A case study from Lefkosia (Nicosia) Cyprus.

The different types of land-use and soil lithology in urban and peri-urban areas of modern cities compose a complex mosaic of soil ecosystems. It is largely unknown how these differences result in changes in bacterial community composition and structure as well as in functional guilds involved in N cycling. To investigate the bacterial composition and the proportion of denitrifiers in agricultural, forested, schoolyard and industrial areas, 24 samples were collected from urban and peri-urban sites of Lefkosia. Bacterial diversity and the proportion of denitrifiers were assessed by NGS and qPCR, respectively. Proteobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Acidobacteria and Planctomycetes were identified as the most dominant phyla across all sites, while agricultural sites exhibited the highest bacterial diversity. Heavy metals such as Co, Pb, V and Al were identified as key factors shaping bacterial composition in industrial and schoolyard sites, while the bacterial assemblages in agricultural and forested sites were associated with Ca. Variance partitioning analysis showed that 10.2% of the bacterial community variation was explained by land use management, 5.1% by chemical elements due to soil lithology, and 1.4% by sampling location. The proportion of denitrifiers varied with land use management. In industrial and schoolyard sites, the abundance of the nosZII bacterial community increased while nirK abundance declined. Our data showed that land use and lithology have a moderate impact on the bacterial assemblages in urban and peri-urban areas of Lefkosia. As the nosZII bacterial community is important to the N2O sink capacity of soils, it would be interesting to elucidate the factors contributing to the proliferation of the nosZII clade in these soils.

The effect of chemical and organic N inputs on N2O emission from rain-fed crops in Eastern Mediterranean.

Nitrogen has a significant contribution to global warming and its reduction in agriculture is expected to reduce N2O emissions having however adverse effects on the productivity of agricultural ecosystems. Maintaining systems productivity with alternative N sources i.e manure and composts could be a strategy also to mitigate N2O emissions. In this paper, we present the effect of different N sources (organic and chemical) on field N2O emissions and how these emissions are associated with soil available N forms (NH4+ and NO3-) in three different rain-fed crops namely barley, pea and vetch grown in Cyprus for two growing seasons. The daily emissions ranged from -3.11 to 12.3 g N-N2O/ha/day, while cumulative emissions ranged from 119 g N-N2O/ha to 660 g N-N2O/ha depending on crop and nitrogen source type. The emissions showed a seasonal pattern and WFPS has been identified as a critical soil parameter controlling daily N2O emissions. The daily N2O fluxes in the current study derives mainly from nitrification irrespectively crop type or nitrogen source type. Specific emission factors for each crop cultivated under different N source type were calculated and ranged from 0.03% ± 0.02-0.34% ± 0.09. The application of manure and chemical fertilizers cause similar intensity of N2O emissions while compost exhibited the lower emission factors. These findings suggest that composts could be integrated in a nutrient management strategy of rain-fed crops with less N2O emissions. The high background emissions found suggest also that other factors than external inputs are associated with N2O emissions and further studies including the response of microbial community structure and their contribution and association with N2O emissions.

Valorization of agricultural wastes could improve soil fertility and mitigate soil direct N2O emissions.

The emerging need for sustainable management of the increasing quantities of urban and industrial organic wastes creates opportunities for the development of alternative strategies for the improvement of degraded soils. The current study was performed to examine the effects of agricultural wastes application on soil bacterial community as well as CO2 and N2O direct gas emissions. Untreated soils were compared with soils, which received the same amount of N (100 µg/g soil) in the form of ammonium nitrate and organic agricultural waste. In particular, soils were incubated with three different organic agricultural wastes, orange (OP), mandarin (MP) and banana peels (BP) and ammonium nitrate (F) after adjusting soil water at 70% of its holding capacity. In the current study, soil chemical characteristics, quantitative PCR of denitrifiers (nirK, nirS, nosZI and nosZII) and16s rRNA amplicon sequencing were assessed to examine the links between the soil microbial communities and short-term soil direct N2O emissions when treated with agricultural wastes. The highest soil direct N2O emissions were recorded in soils received ammonium nitrate while soils received agricultural wastes exhibited substantially lower soil direct N2O emissions. On the contrary, agricultural wastes stimulated CO2 accumulation as well as the growth of copiotrophic bacterial groups like Proteobacteria and Firmicutes. Interestingly, direct soil N2O emissions were decoupled from the density of denitrifier community while agricultural wastes caused a substantial reduction of the relative abundance of bacterial taxa associated with N2O emissions in the soil. This study proves evidence that agricultural wastes could be integrated in a waste management strategy, which inter alia includes their direct use in agricultural ecosystems resulting in reduced N2O emissions.

Optimizing Resource Allocation in a Cowpea (Vigna unguiculata L. Walp.) Landrace Through Whole-Plant Field Phenotyping and Non-stop Selection to Sustain Increased Genetic Gain Across a Decade.

Cowpea is a warm-season legume, often characterized as an orphan or underutilized crop, with great future potential, particularly under the global change. A traditional cowpea landrace in Cyprus is highly valued for fresh pod consumption in the local cuisine. In order to improve the yield potential of the landrace, the long-term response to direct selection for fresh pod yield and the associated changes in fodder and root biomass were investigated in a variety of fertility regimes under real field conditions. The non-stop selection process employed comprehensive pod, fodder, and root phenotyping at the level of the individual plant and resulted in the creation of a range of highly improved sibling lines with differential adaptation to micro-environments and with an improved ratio of pod to shoot and root biomass. The average rate of increase per year for fresh pod yield is at the level of 180 g per plant despite the relatively narrow genetic base of a single landrace and it is seemingly inexhaustible testifying to the great plasticity of the cowpea genome and the potential of the methodology to capture it. The corresponding high genetic gain was also confirmed under dense stands where the difference in pod yield between the best selection and the control amounted to 31.37%. Thus, the new focus apart from the simple variety maintenance should also include the continuous improvement and exploitation of micro-adaptation processes specific for individual fields that allow quick responses to environmental and climatic changes. This work presents also a novel approach to the multiple challenges encountered in root phenotyping and a method to meaningfully associate it with whole-plant performance in field conditions.

The effects of different soil nutrient management schemes in nitrogen cycling.

It is imperative for sustainable agriculture to explore practices and inputs creating low N2O emission capacity without reducing the productivity of the agricultural system. To evaluate different nutrient management schemes, a microcosm study was conducted to assess the direct N2O emission from soil. Four different treatments were used to provide a preliminary assessment of N2O emissions, as well as the concentrations of nitrates (NO3-) and ammonium (NH4+) produced in soil: compost (derived from green plant residues), chickpea residues (green manure) in two different N concentrations (2.6% and 5.5%, respectively) and ammonium nitrate (fertilizer). The soil was thoroughly mixed with the organic amendments and ammonium nitrate and incubated for 31 days. The emissions of N2O were higher in green manure with high-N content, as a source of nitrogen in the soil, and were similar to the emissions measured from the chemically fertilized soil. In particular, chickpea residues, with high-N content, exhibited cumulative N2O emissions, equal to 266.17 µg N/m2, whereas in fertilized soil the emissions were 267.10 µg N/m2. On the contrary, the incorporation of chickpea plant residues with low-N content can be an efficient way to minimize the N2O emissions at 21.63 µg N/m2. The emissions of N2O when compost was applied, remained relatively low, equal to 5.47 µg N/m2, and in comparison to soil without any treatment. Overall, a positive association between NH4+, NO3- in soil and N2O emissions were observed. However, this response was treatment depended, and the significant positive correlation between NH4+ and N2O emissions were noticed in soils treated with ammonium nitrate, chickpea residues with low N content, as well as untreated controls. On the contrary, the positive correlation observed between NO3- and N2O emissions in soils receiving compost and high N chickpea residues, suggest that the different treatments are differentially affecting the processes that are contributing to N2O emissions in agricultural soils. These findings, emphasize that the different nutrient management schemes are differentially affecting the main process contributing to N2O emissions in agricultural soils.

Converting environmental risks to benefits by using spent coffee grounds (SCG) as a valuable resource.

Coffee is perhaps one of the most vital ingredients in humans' daily life in modern world. However, this causes the production of million tons of relevant wastes, i.e., plastic cups, aluminum capsules, coffee chaff (silver skin), and spent coffee grounds (SCG), all thrown untreated into landfills. It is estimated that 1 kg of instant coffee generates around 2 kg of wet SCG; a relatively unique organic waste stream, with little to no contamination, separated directly in the source by the coffee shops. The produced waste has been under researchers' microscope as a useful feedstock for a number of promising applications. SCG is considered a valuable, nutrients rich source of bioactive compounds (e.g., phenolics, flavonoids, carotenoids, lipids, chlorogenic and protocatechuic acid, melanoidins, diterpenes, xanthines, vitamin precursors, etc.) and a useful resource material in other processes (e.g., soil improver and compost, heavy metals absorbent, biochar, biodiesel, pellets, cosmetics, food, and deodorization products). This paper aims to provide a holistic approach for the SCG waste management, highlighting a series of processes and applications in environmental solutions, food industry, and agricultural sector. Thus, the latest developments and approaches of SCG waste management are reviewed and discussed.

Bioaugmentation of thiabendazole-contaminated soils from a wastewater disposal site: Factors driving the efficacy of this strategy and the diversity of the indigenous soil bacterial community.

The application of the fungicide thiabendazole (TBZ) in fruit packaging plants (FPP) results in the production of effluents which are often disposed in adjacent field sites. These require remediation to prevent further environmental dispersal of TBZ. We assessed the bioaugmentation potential of a newly isolated TBZ-degrading bacterial consortium in a naturally contaminated soil (NCS) exhibiting a natural gradient of TBZ levels (12000, 400, 250 and 12 mg kg-1). The effect of aging on bioaugmentation efficacy was comparatively tested in a soil with similar physicochemical properties and soil microbiota, which was artificially, contaminated with the same TBZ levels (ACS). The impact of bioaugmentation and TBZ on the bacterial diversity in the NCS was explored via amplicon sequencing. Bioaugmentation effectively removed TBZ from both soils at levels up to 400 mg kg-1 but failed at the highest contamination level (12000 mg kg-1). Dissipation of TBZ in bioaugmented samples showed a concentration-dependent pattern, while aging of TBZ had a slight effect on bioaugmentation efficiency. Bioaugmentation had no impact on the soil bacterial diversity, in contrast to TBZ contamination. Soils from the hotspots of TBZ contamination (12000 mg kg-1) showed a drastically lower α-diversity driven by the dominance of ß- and γ-proteobacteria at the expense of all other bacterial phyla, especially Actinobacteria. Overall, bioaugmentation with specialized microbial inocula could be an effective solution for the recovery of disposal sites contaminated with persistent chemicals like TBZ.

Isolation of a bacterial consortium able to degrade the fungicide thiabendazole: the key role of a Sphingomonas phylotype.

Thiabendazole (TBZ) is a fungicide used in fruit-packaging plants. Its application leads to the production of wastewaters requiring detoxification. In the absence of efficient treatment methods, biological depuration of these effluents could be a viable alternative. However, nothing is known regarding the microbial degradation of the recalcitrant and toxic to aquatics TBZ. We report the isolation, via enrichment cultures from a polluted soil, of the first bacterial consortium able to rapidly degrade TBZ and use it as a carbon source. Repeated efforts using various culture-dependent approaches failed to isolate TBZ-degrading bacteria in axenic cultures. Denaturating gradient gel electrophoresis (DGGE) and cloning showed that the consortium was composed of α-, ß- and γ-Proteobacteria. Culture-independent methods including antibiotics-driven selection with DNA/RNA-DGGE, q-PCR and stable isotope probing (SIP)-DGGE identified a Sphingomonas phylotype (B13) as the key degrading member. Cross-feeding studies with structurally related chemicals showed that ring substituents of the benzimidazole moiety (thiazole or furan rings) favoured the cleavage of the imidazole moiety. LC-MS/MS analysis verified that TBZ degradation proceeds via cleavage of the imidazole moiety releasing thiazole-4-carboxamidine, which was not further transformed, and the benzoyl moiety, possibly as catechol, which was eventually consumed by the bacterial consortium as suggested by SIP-DGGE.

Glucosinolate biosynthesis in Eruca sativa.

Glucosinolates (GSLs) are a highly important group of secondary metabolites in the Caparalles order, both due to their significance in plant-biome interactions and to their chemoprotective properties. This study identified genes involved in all steps of aliphatic and indolic GSL biosynthesis in Eruca sativa, a cultivated plant closely related to Arabidopsis thaliana with agronomic and nutritional value. The impact of nitrogen (N) and sulfur (S) availability on GSL biosynthetic pathways at a transcriptional level, and on the final GSL content of plant leaf and root tissues, was investigated. N and S supply had a significant and interactive effect on the GSL content of leaves, in a structure-specific and tissue-dependent manner; the metabolites levels were significantly correlated with the relative expression of the genes involved in their biosynthesis. A more complex effect was observed in roots, where aliphatic and indolic GSLs and related biosynthetic genes responded differently to the various nutritional treatments suggesting that nitrogen and sulfur availability are important factors that control plant GSL content at a transcriptional level. The biological activity of extracts derived from these plants grown under the specific nutritional schemes was examined. N and S availability were found to significantly affect the cytotoxicity of E. sativa extracts on human cancer cells, supporting the notion that carefully designed nutritional schemes can promote the accumulation of chemoprotective substances in edible plants.

Dissipation, metabolism and sorption of pesticides used in fruit-packaging plants: Towards an optimized depuration of their pesticide-contaminated agro-industrial effluents.

Wastewaters from the fruit-packaging industry constitute a serious point source contamination with pesticides. In the absence of effective depuration methods, they are discharged in municipal wastewater treatment plants or spread to land. Modified biobeds could be an applicable solution for their treatment. We studied the dissipation of thiabendazole (TBZ), imazalil (IMZ), ortho-phenylphenol (OPP), diphenylamine (DPA) and ethoxyquin (EQ), used by the fruit-packaging industry, in anaerobically digested sewage sludge, liquid aerobic sewage sludge and in various organic substrates (biobeds packing materials) composed of soil, straw and spend mushroom substrate (SMS) in various volumetric ratios. Pesticide sorption was also determined. TBZ and IMZ showed higher persistence especially in the anaerobically digested sewage sludge (DT50=32.3-257.6d), in contrast to OPP and DPA which were rapidly dissipated especially in liquid aerobic sewage sludge (DT50=1.3-9.3d). EQ was rapidly oxidized mainly to quinone imine (QI) which did not persist and dimethyl ethoxyquinoline (EQNL, minor metabolite) which persisted for longer. Sterilization of liquid aerobic sewage sludge inhibited pesticide decay verifying the microbial nature of pesticide dissipation. Organic substrates rich in SMS showed the highest dissipation capacity with TBZ and IMZ DT50s of ca. 28 d compared to DT50s of >50 d in the other substrates. TBZ and IMZ showed the highest sorption affinity, whereas OPP and DPA were weakly sorbed. Our findings suggest that current disposal practices could not guarantee an efficient depuration of effluents from the fruit-packaging industry, whereas SMS-rich biobed organic substrates show efficient depuration of effluents from the fruit-packaging industry via accelerated dissipation even of recalcitrant fungicides.

Detection and geographical distribution of the organophosphate resistance-associated Δ3Q ace mutation in the olive fruit fly, Bactrocera oleae (Rossi).

BACKGROUND: The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the most important pest of olives. Its control is based mostly on organophosphate (OP) insecticides, a practice that has led to resistance development. OP resistance in B. oleae has been associated with three mutations in the acetylcholinesterase (AChE), the product of ace gene. The current study presents new diagnostic tests for the detection of the ace mutations and aims at monitoring the frequency of the Δ3Q mutation, which appears associated with resistance at higher OP doses in natural olive fly populations. RESULTS: An allele-specific polymerase chain reaction (PCR), a PCR-RFLP (restriction fragment length polymorphism) and a Taq-Man test were developed for the Δ3Q mutation detection and a new duplex quantitative PCR assay was designed for the G488S and I214V mutations. Moreover, the frequency of Δ3Q mutation was examined in ten populations of eight countries around the Mediterranean basin. The highest frequencies (10%) were found in Greece and Italy, whereas a gradual decrease of Δ3Q frequency towards the western Mediterranean was noted. CONCLUSION: Robust tests for insecticide resistance mutations at their incipient levels are essential tools to monitor the increase and geographical spread of such mutations. Three different tests were developed for AChE-Δ3Q that indicated its association with OP applications across the Mediterranean.

The impact of biofumigation and chemical fumigation methods on the structure and function of the soil microbial community.

Biofumigation (BIOF) is carried out mainly by the incorporation of brassica plant parts into the soil, and this fumigation activity has been linked to their high glucosinolate (GSL) content. GSLs are hydrolyzed by the endogenous enzyme myrosinase to release isothiocyanates (ITCs). A microcosm study was conducted to investigate the effects induced on the soil microbial community by the incorporation of broccoli residues into soil either with (BM) or without (B) added myrosinase and of chemical fumigation, either as soil application of 2-phenylethyl ITC (PITC) or metham sodium (MS). Soil microbial activity was evaluated by measuring fluorescein diacetate hydrolysis and soil respiration. Effects on the structure of the total microbial community were assessed by phospholipid fatty acid analysis, while the impact on important fungal (ascomycetes (ASC)) and bacterial (ammonia-oxidizing bacteria (AOB)) guilds was evaluated by denaturating gradient gel electrophoresis (DGGE). Overall, B, and to a lesser extent BM, stimulated microbial activity and biomass. The diminished effect of BM compared to B was particularly evident in fungi and Gram-negative bacteria and was attributed to rapid ITC release following the myrosinase treatment. PITC did not have a significant effect, whereas an inhibitory effect was observed in the MS-treated soil. DGGE analysis showed that the ASC community was temporarily altered by BIOF treatments and more persistently by the MS treatment, while the structure of the AOB community was not affected by the treatments. Cloning of the ASC community showed that MS application had a deleterious effect on potential plant pathogens like Fusarium, Nectria, and Cladosporium compared to BIOF treatments which did not appear to inhibit them. Our findings indicate that BIOF induces changes on the structure and function of the soil microbial community that are mostly related to microbial substrate availability changes derived from the soil amendment with fresh organic materials.

Impact of nitrogen and sulfur fertilization on the composition of glucosinolates in relation to sulfur assimilation in different plant organs of broccoli.

Broccoli (Brassica oleracea var. italica) is one of the most important winter season vegetables and a rich source of chemoprotective molecules, including glucosinolates (GSL). The aim of this study was to investigate the impact of nitrogen (N) and sulfur (S) fertilization on GSL concentration and composition in different parts of broccoli plants. A greenhouse experiment was performed, with four different treatments of sulfur (10, 30, 70, and 150 kg/ha) and three treatments of nitrogen (50, 250, and 600 kg/ha). GSL concentrations and plant growth responded to the N supply, but this was not observed above the 250 kg N/ha dose. On the contrary, plant growth did not respond to the S supply, whereas GSL concentrations showed a sharp response to the whole range of S applications (from 10 to 150 kg/ha). Glucosinolate composition was altered differentially in the examined plant parts. Aliphatic GSL were more abundant in the florets and leaves, whereas indolyl GSLs were dominant in roots, in which aromatic GSL were also observed. High nitrogen fertilization had a higher impact on indolyl compared to aliphatic GSLs concentration. More importantly, a high concentration of aliphatic GSL, >2.4 micromol/g dry weight (dw), and high S assimilation into aliphatic GSL were consistently observed in the florets compared to other broccoli parts, indicating adaptable processes for nitrogen and sulfur regarding synthesis and transport of aliphatic GSL for these organs.

Microwave-assisted extraction of glucosinolates from Eruca sativa seeds and soil: comparison with existing methods.

INTRODUCTION: Glucosinolates (GSLs) are secondary plant metabolites that are abundant in brassicas and their hydrolysis products, isothiocyanates, are toxic to soil pathogens. Efficiency and extraction time are critical for routine analysis of GSLs in plant tissues. Robust analytical procedures are required for the extraction of GSL from soil. OBJECTIVE: Development and optimisation of a microwave-assisted extraction (MAE) method for the recovery of GSL from plant tissues and soil and comparison of its efficiency with other established extraction methods. METHODOLOGY: Solvents, temperature, microwave power and extraction time were examined as parameters controlling MAE efficiency. In rocket seeds the efficiency of MAE was determined through recovery of GSLs from seeds and of sinigrin (1) that was used as internal standard. MAE was then compared with the certified ISO-9167 method and an ultrasonic-assisted extraction (UAE). MAE was also applied for the extraction of GSLs from soils fortified with 1 at three fortification levels. The efficiency of MAE was compared with a recently proposed agitation-filtration (AGIT) extraction method. RESULTS: The optimum conditions identified for extraction of GSLs from seeds were: methanol extraction at 250 W and 80 degrees C for 10 min. MAE and ISO methods showed comparable efficiencies and higher than UAE. In soil, both methods resulted in nearly 100% recovery of 1 at all fortification levels, although MAE achieved this recovery after a single extraction step compared with AGIT, which required two. CONCLUSIONS: The MAE developed is a simple and rapid method for the extraction of GSLs from plant tissues and soil that can be applied to a large number of samples, thus reducing the time of analysis.