Antonio Gelsomino

Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis.

Bacterial community structure was studied in a Flevo silt loam (FSL) soil microplot, as well as in 15 other soils, by using DNA extraction followed by molecular fingerprinting. Total community DNA was extracted and purified by a direct method, which yielded amplifiable DNA of high molecular weight for all soils. A variable region of the 16S rRNA gene was then amplified by PCR with bacterial primers, resulting in a mixture of amplicons separable via denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of FSL soil were indicative of dominant soil bacterial types, as evidenced by assessing the amplification of Enterobacter cloacae and Arthrobacter sp. targets in a soil DNA background. These targets produced barely detectable bands when present in soil DNA at roughly 5 x 10(6) genome equivalents per g dry soil, and strong bands at 27-fold higher levels. The PCR-DGGE analysis of the FSL soil was highly reproducible. Furthermore, different single versus composite topsoil samples yielded similar DGGE profiles with respect to major bands. In addition, samples taken along vertical soil cores (0-45 cm depth) revealed relative stability of the DGGE profiles. The profiles produced with DNA obtained from different aggregate size fractions of this soil were also similar with respect to the main bands. Moreover, FSL topsoil samples taken over a 1-year period (fallow soil) yielded stable profiles. These data suggested that the soil bacterial communities thus determined were dominated by a limited number of stable and ubiquitous types. The 16 soils, representing varying types and geographical locations, were assessed for differences in their bacterial DGGE profiles. There were striking differences between the profiles obtained for these soils. Evidence was found for the hypothesis that similar soil types tend to contain similar structures of the dominating bacterial types as revealed by the DGGE profiles.

Effects of compost addition and simulated solarisation on the fate of Ralstonia solanacearum biovar 2 and indigenous bacteria in soil

Abstract The effects of compost addition and simulated solarisation of soil on the survival of Ralstonia solanacearum biovar 2 strain 1609, as well as on the structure of indigenous soil bacterial communities, were analysed. In addition, effects on the invasion of susceptible test plants by strain 1609 were assessed. In untreated soil in microcosms and the field, strain 1609 showed slow progressive declines, from 10(6)-10(7) to roughly 10(4)-10(5) CFU per g dry soil in around 60 days. When these soils were used in suppressiveness tests, a majority of plants developed symptoms of wilting and revealed the presence of the pathogen in their lower stem parts, as evidenced by immunofluorescence colony staining (IFC) and polymerase chain reaction (PCR). Solarisation of unamended soil did not drastically affect R. solanacearum survival or plant invasiveness. However, the addition of household compost resulted in enhanced R. solanacearum population decline rates, as well as reduced numbers of diseased plants in suppressiveness tests. Combined solarisation and compost addition yielded differential results between microcosms and the field. Some healthy-looking plants, primarily from soils treated with compost, revealed the latent presence of strain 1609 in the lower stem parts. The eubacterial and beta-subgroup proteobacterial communities in the differentially treated soil microcosms were rather stable, as evidenced by analysis of PCR-denaturing gradient gel electrophoresis (DGGE) generated molecular profiles. However, compost amendment clearly induced changes in these communities, which were detectable until the end of the experiment; two major bands, affiliated with Variovorax paradoxus and Aquaspirillum psychrophylum, were associated with the compost amendment. The decrease in abundance of R. solanacearum in the compost-amended soils was confirmed by the DGGE profiles.

Biochemical characterization of soil organic compounds extracted by 0.5 m K2SO4 before and after chloroform fumigation

Abstract The organic C (EC), total N (EN), anthrone-, phenol- and orcinol-reactive C (ARC, PRC and ORC, respectively), ninhydrin-reactive N (NRN), Folin-Ciocalteus reagent-reactive C and N compounds (FRC and FRN) and deoxyribose containing compounds (diphenylamine-reactive compounds, DRC) extracted by 0.5 m K2SO4 before (non-fumigated extracts) and after (fumigated extracts) CHC13 fumigation were evaluated in 8 very different soils. All quantities were significantly linearly correlated between them and with total organic C (TOC) and total N (TN) as well. The frequency distribution of the significance levels passing from non-fumigated to fumigated extracts and γ-values (fumigated minus non-fumigated values for the same class of compounds) showed a shift towards the highest significances; this may indirectly confirm the action of chloroform in lysing soil microbial cells. The significance levels (P) of TOC correlated vs fumigated extracts were higher than vs non-fumigated extracts and γ -values, thus indicating a release of non-biomass C organic compounds after CHCl3 fumigation. Since TN vs fumigated extracts showed significance levels higher than non-fumigated extracts but equal to γ-values, any release of non-biomass N probably did not occur. These considerations were confirmed by multiple regression analysis when EC and EN (dependent variables) were reconstituted taking into account TOC and TN as additional independent variables, respectively. However, released non-biomass C organic compounds (likely sugars) compared to TOC were very low and could be considered to be negligible. A low release of ribose-containing compounds (ORC) occurred, thus indicating that the amount of RNA material released after CHCl3 exposure was negligible. Deoxyribose-containing compounds rendered extractable after CHCl3 fumigation were probably derived from bacterial plasmids rather than from bacterial and eukaryotic chromosomes. The possibility of characterizing soil microbial biomass C and N compounds by adopting suitable colorimetric methods, as demonstrated in this work, is a further advantage of the fumigation-extraction method when compared with the fumigation-incubation method.

Multiresidue analysis of pesticides in fruits and vegetables by gel permeation chromatography followed by gas chromatography with electron-capture and mass spectrometric detection

Abstract A wide range screening method for multiresidue analysis of seventy-seven pesticides (twelve organohalogens, forty-five organonitrogens, eleven organophosphorus and nine pyrethroids) in agricultural products is proposed. Pesticide residues were extracted from crop samples with acetone followed by dichloromethane partitioning. Crop extracts were cleaned-up by gel permeation chromatography equipped with a 10 mm diameter column. Analytical screening was by gas chromatography using long, narrow-bore fused-silica open-tubular columns equipped with electron-capture detection (ECD). Recoveries of majority of pesticides from spiked samples of carrot, melon and tomato at fortification levels of 0.04–0.10 mg/kg ranged from 70 to 108%. The lowest recovery was for chlormephos (51.5%). Limits of detection were less than 0.01 mg/kg for ECD. Confirmation of pesticide identity was performed by gas chromatography-mass spectrometry in selected-ion monitoring mode. The multiresidue procedure was applied in routine crop analysis: a 9-month period data are reported.

Effect of nitrate and humic substances of different molecular size on kinetic parameters of nitrate uptake in wheat seedlings

The kinetic parameters of nitrate uptake (Imax, Km and Cmin) were evaluated in young seedlings of Triticum durum L., cv. Appulo, exposed to nitrate and/or to soil-extracted humic acids (HAs) of different molecular weight. The uptake was enhanced after induction at low levels of nitrate (50 μM KNO3), while it was inhibited after induction at higher concentrations (2000 μM). The kinetic parameters of uptake were selectively influenced by pre-treatment with HAs: total (TE) and, at a greater extent, low (LMS,  3500 Da)-treated plants. An additive effect was shown when nitrate and humic substances were provided simultaneously: the uptake rate was enhanced in TE- and LMS-treated plants, but was strongly delayed in HMS-treated plants. Removal of nitrate and/or humic fractions de-induced the system and NO3— uptake rate decreased. Exposure to HAs was not able to induce nitrate reductase activity in root and leaf tissues. Inhibitors of protein synthesis p-fluorophenylalanine and cycloheximide reversed the positive effect of LMS fraction on nitrate uptake. This would support the hypothesis of a promoting effect of HAs on the molecular expression of proteins of the nitrate transport system. Wirkung von Nitrat und Humusstoffen unterschiedlicher Molekulgrose auf kinetische Parameter der Nitrataufnahme bei Weizenkeimpflanzen Die kinetischen Charakteristika der Nitrataufnahme von Hartweizenkeimpflanzen wurden nach Behandlung mit Nitratlosung ohne und mit Humusstoffen unterschiedlicher Molekulgrose ermittelt. Bereits bei dem niedrigsten Nitratangebot wurde die Nitrataufnahme induziert, wohingegen sie bei 2000 μM KNO3 nach Erreichen des Induktionsmaximums gehemmt wurde. Humusstoffe mit geringer Molekulgrose erhohten die Nitrataufnahmerate und die Effizienz des Transportsystems, erkennbar durch niedrige kM- und Cmin-Werte. Die Kombination von Nitrat und niedermolekularen Humusstoffen erhohte ebenfalls die Nitrataufnahme, wohingegen Pflanzen, die mit hochmolekularen Verbindungen behandelt wurden, Nitrat verzogert aufnahmen. Die Entfernung von Nitrat und/oder Humusstoffen de-induzierte die Nitrataufnahme, wobei die Nitrataufnahme auf ein Niveau fiel, das niedriger als das Ausgangsniveau war. Die Behandlung mit Humusstoffen allein hat keine Nitratreduktaseaktivitat in der Wurzel oder im Blattgewebe induziert. Hemmstoffe der Proteinbiosynthese kehrten den positiven Effekt niedermolekularer Humusstoffe um. Die Ergebnisse werden hinsichtlich einer Interaktion von Humusstoffen und der Expression des Nitrattransportproteins diskutiert.

ADSORPTION OF TWO QUINOLINECARBOXYLIC ACID HERBICIDES ON HOMOIONIC MONTMORILLONITES

The adsorption of the herbicides quinmerac 7-chloro-3-methylquinoline-8-carboxyl ic acid (QMe) and quinclorac 3,7-dichloroquinoline-8-carboxylic acid (QCl) on homoionic Fe3+-, Al3+-, Cu2+-, Ca2+-, K+- and Na+-exchanged montmorillonite was studied in aqueous solution. Adsorption data were fitted to the logarithmic form of the Freundlich equation. Ca- and Na-exchanged montmorillonites were ineffective in the adsorption of QMe. On the other hand, the QMe adsorption on Fe-exchanged montmorillonite was rapid and the equilibrium was attained after 15 min. An H-type isotherm was observed for the QMe adsorption on Fe-clay, indicating a high affinity of the solute for the sorption sites and almost complete adsorption from dilute solution. On the other hand, the adsorption isotherm of QMe on Al- and K-clay was of the S-type. This shape suggests that the solvent molecules may compete for the sorption sites. A Fourier transform infrared (FTIR) study suggested that the adsorption mechanism of QMe on Fe-, Al- and K-clay involves the protonation of QMe molecule due to the acidic water surrounding the saturating cations. The greater acidity of Fe-clay compared with Al- and K-clay explains both the lower QMe adsorption observed on Al and K systems and the lack of adsorption on Na and Ca systems. In contrast, the formation of a Cu complex permitted QMe to be adsorbed to a large extent to Cu-clay as shown by FTIR analysis. The QCl was adsorbed only by Fe-clay and the adsorption isotherm of QCl on Fe clay was of the S-type. This finding is consistent with the lower basic character of the QCl molecule nitrogen. In fact, the replacement of the electron-releasing methyl group in QMe with an electron-withdrawing Cl atom to form QCl makes the nitrogen lone-pair electrons of the quinoline ring unavailable for either protonation or complexation.

ADSORPTION MECHANISMS OF IMAZAMETHABENZ-METHYL ON HOMOIONIC MONTMORILLONITE

The adsorption of the herbicide imazamethabenz-methyl, a mixture of the two isomers methyl (±)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1 H-imidazol-2-yl]-4-methylbenzoate para isomer) and methyl (±)-2-[4,5-dihydro-4-methyl-4-(l-methylethyl)-5-oxo-1 H-imidazol-2-yl]-5-methyl-benzoate (meta isomer), from water onto Al3+, Fe3+-, Ca2+-, K+- and Na+-montmorillonite was studied by analytical (HPLQ methods. The adsorption from an organic solvent was also investigated by spectroscopic (IR) and X-ray diffraction measurements. It was observed that, depending on the acidic properties of the exchangeable cations, two different mechanisms may take place. The first one, acting on Fe3+- and Al3+-clays, involves the protonation of the more basic nitrogen atom of imidazolinone ring of the herbicide because of a proton transfer from the acidic metal-bound water, followed by adsorption on the clay surfaces. In this case, the clay surfaces have greater affinity for the meta than the para isomer, due to the extra-stabilization of the meta protonated form by resonance. The second mechanism, taking place on Ca2+-, K+- and Na+-clays, is hydrogen-bond formation between the ester carbonyl group of the herbicide and hydration water metal ions and is not affected by the structure of the isomers.

Compost from Fresh Orange Waste: A Suitable Substrate for Nursery and Field Crops?

Composting represents a valuable strategy for recycling orange processing waste for use as soil conditioner, provided compost maturity is duly evaluated. Following a 5-month aerobic bioconversion, orange waste reached an acceptable degree of maturity in terms of humification parameters, absence of phytotoxicity (determined on test plants according to ISO methods) and low content of simple phenolic compounds. A greenhouse experiment with two nursery crops (tomato and zucchini) showed that, depending on the characteristics of the growing substrate (vermiculite or perlite), orange compost addition selectively induced pH and electrical conductivity (EC) increases, which in turn would affect plant growth responses. In field crops shoot mass weight was increased after compost addition. Results suggest that at field scale, orange compost may be used for organic fertilization; while in nursery crops it can be mixed with commercial potting substrates selected in relation to plant sensitivity. In horticultural crops, for its greater ability to buffer pH and EC changes, vermiculite could represent the preferred complementary substrate compared to perlite.

AN ATTEMPT TO MODEL THE INDUCTION AND FEEDBACK INHIBITION OF NITRATE UPTAKE IN WHEAT SEEDLINGS

The aim of the present study was to develop a mathematical model in attempt to describe the induction and feedback inhibition phases of nitrate uptake in wheat (Triticum durum L. cv. Appulo) seedlings. The model proposed is based on three autonomous, non-linear, and time-dependent differential equations showing good agreement with our experimental data. The model could be fruitfully applied for a correct evaluation of different patterns of nitrate uptake owing to genetic and physiometabolic plant characteristics and external nutrient availability.

Artemisia arborescens L. leaf litter: phytotoxic activity and phytochemical characterization

Artemisia arborescens L. is a perennial fast-growing Mediterranean shrub, which releases abundant leaf litter upon soil surface throughout the year. The paper aimed to both evaluate the phytotoxic potential and identify major compounds occurring in the plant leaf litter. Following methanolic maceration of the leaf litter, the crude extract was then sequentially extracted with hexane, chloroform and ethyl acetate through a bio-guided fractionation method. The phytotoxic potential of the methanolic extract and its solvent fractions was assessed in vitro on germination and root growth of two sensitive (Lactuca sativa L., Raphanus sativus L.) and native (Amaranthus retroflexus L., Cynodon dactylon (L.) Pers.) species. Moreover, the most active fractions were chemically characterized by GC–MS and HPTLC analysis. In all species, the physiological processes were highly inhibited by both the methanolic extract and its solvent fractions. Several classes of biologically active phytochemicals such as terpenoids, fatty acids, lignans and phenolic compounds were identified in all fractions. Artemisia arborescens leaf litter could be considered an important source of biologically active phytochemicals, which may have a significant allelopathic impact towards neighbouring species once released into the environment.