Exploration of the anthocyanin and proanthocyanidin profile of Greek red grape skins belonging to Vradiano, Limnio, and Kotsifali cultivars, analyzed by a novel LC-QTOF-MS/MS method.

INTRODUCTION: Winegrape varieties Kotsifali, Limnio, and Vradiano OBJECTIVE: The aim of this study was to develop a liquid chromatographic quadrupole time-of-flight tandem mass spectrometric (LC-QTOF-MS/MS) method for the investigation of the anthocyanin and proanthocyanidin content of Greek grape varieties employing target and suspect screening strategies. METHODOLOGY: A novel LC-QTOF-MS/MS method was developed and validated to assess the anthocyanin content of Kotsifali, Limnio, and Vradiano grape varieties. Sixteen grape samples were collected from the main growing areas of each variety in Greece. The influence of the grape variety on the anthocyanin and proanthocyanidin composition of three Greek winegrapes was investigated using chemometrics. RESULTS: Excellent linearity (R2 > 0.99) was achieved for all the target analytes, and recoveries ranged between 90.1% and 119.1%. The limits of quantification (LOQs) and limits of detection (LODs) were calculated over the range of 0.020-0.40 mg/g and 0.010-0.13 mg/g, respectively. The RSD% was lower than 9.1% and 7.3% for intra-day and inter-day studies, respectively, indicating satisfactory trueness and precision. Target and suspect screening resulted in the identification of 5 and 26 anthocyanins, respectively. CONCLUSIONS: Kotsifali variety exhibited a higher concentration of anthocyanins compared with Vradiano and Limnio. Higher levels of mean degree of polymerization (mDp) and different percentage levels of prodelphinidins (%P) were established among the varieties.

Investigation of Rosa species by an optimized LC-QTOF-MS/MS method using targeted and non-targeted screening strategies combined with multivariate chemometrics.

INTRODUCTION: Plants of the Rosa genus are renowned for their pronounced and pleasant aroma and colors. OBJECTIVE: The aim of this work was to develop a novel liquid chromatographic triple quadrupole time-of-flight tandem mass spectrometric (LC-QTOF-MS/MS) method for the investigation of the bioactive fingerprint of petals of different genotypes belonging to Rosa damascena and Rosa centifolia species. METHODOLOGY: Central composite design (CCD) of response surface methodology (RSM) was used for the optimization of the LC-QTOF-MS/MS method. The method was validated and target, suspect, and non-target screening workflows were applied. Statistical analysis and chemometric tools were utilized to explore the metabolic fingerprint of the Rosa species. RESULTS: RSM revealed that the optimal extraction parameters involved mixing 11 mg of sample with 1 mL of MeOH:H2O (70:30, v/v). Target analysis confirmed the presence of 11 analytes, all of which demonstrated low limits of quantification (LOQs; as low as 0.048 ng mg-1) and sufficient recoveries (RE: 85%-107%). In total, 28 compounds were tentatively identified through suspect analysis. Non-target analysis enabled the generation of robust OPLS-DA and HCA models that classified the samples according to their species with 100% accuracy. CONCLUSIONS: A novel LC-QTOF-MS/MS method was developed and applied in the analysis of 47 R. centifolia and R. damascena flowers belonging to different genotypes.

Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

Nanocapsules of ZnO Nanorods and Geraniol as a Novel Mean for the Effective Control of Botrytis cinerea in Tomato and Cucumber Plants.

Inorganic-based nanoparticle formulations of bioactive compounds are a promising nanoscale application that allow agrochemicals to be entrapped and/or encapsulated, enabling gradual and targeted delivery of their active ingredients. In this context, hydrophobic ZnO@OAm nanorods (NRs) were firstly synthesized and characterized via physicochemical techniques and then encapsulated within the biodegradable and biocompatible sodium dodecyl sulfate (SDS), either separately (ZnO NCs) or in combination with geraniol in the effective ratios of 1:1 (ZnOGer1 NCs), 1:2 (ZnOGer2 NCs), and 1:3 (ZnOGer2 NCs), respectively. The mean hydrodynamic size, polydispersity index (PDI), and ζ-potential of the nanocapsules were determined at different pH values. The efficiency of encapsulation (EE, %) and loading capacity (LC, %) of NCs were also determined. Pharmacokinetics of ZnOGer1 NCs and ZnOGer2 NCs showed a sustainable release profile of geraniol over 96 h and a higher stability at 25 ± 0.5 °C rather than at 35 ± 0.5 °C. ZnOGer1 NCs, ZnOGer2 NCs and ZnO NCs were evaluated in vitro against B. cinerea, and EC50 values were calculated at 176 µg/mL, 150 µg/mL, and > 500 µg/mL, respectively. Subsequently, ZnOGer1 NCs and ZnOGer2 NCs were tested by foliar application on B. cinerea-inoculated tomato and cucumber plants, showing a significant reduction of disease severity. The foliar application of both NCs resulted in more effective inhibition of the pathogen in the infected cucumber plants as compared to the treatment with the chemical fungicide Luna Sensation SC. In contrast, tomato plants treated with ZnOGer2 NCs demonstrated a better inhibition of the disease as compared to the treatment with ZnOGer1 NCs and Luna. None of the treatments caused phytotoxic effects. These results support the potential for the use of the specific NCs as plant protection agents against B. cinerea in agriculture as an effective alternative to synthetic fungicides.

Coated Cu-doped ZnO and Cu nanoparticles as control agents against plant pathogenic fungi and nematodes.

In the current study, coated copper nanoparticles with polyethylene glycol 8000 (Cu@PEG NPs) and copper-doped zinc oxide nanoparticles with diethylene glycol (Cu-doped ZnO@DEG NPs) have been synthesized via solvothermal and microwave-assisted process, physicochemical characterized, and studied as nano-fungicides and nano-nematicides. Spheroidal Cu-doped ZnO@DEG NPs and urchin-like Cu@PEG NPs have been isolated with average crystallite sizes of 12 and 21 nm, respectively. The Cu doping (11.3 wt%) in ZnO lattice (88.7 wt%) was investigated by Rietveld refinement analysis and confirmed by X-ray Diffraction (XRD) and X-ray Photoelectron Spectroscopy (XPS). The Cu-doped ZnO@DEG and Cu@PEG NPs revealed a growth inhibition of fungi Botrytis cinerea (B. cinerea) and Sclerotinia sclerotiorum (S. sclerotiorum) and nematode paralysis of Meloidogyne javanica in a dose-dependent manner. Cu-doped ZnO@DEG NPs were more effective against M. javanica (EC50 = 2.60 µg/mL) than the Cu@PEG NPs (EC50 = 25 µg/mL). In contrast, the antifungal activity was approximately similar for both NPs, with EC50 values at 310 and 327 µg/mL against B. cinerea, respectively, and 260 and 278 µg/mL against S. sclerotiorum, respectively. Lettuce (Lactuca sativa) plants were inoculated with S. sclerotiorum or M. javanica and sprayed with either Cu-doped ZnO@DEG NPs or Cu@PEG NPs. The antifungal effect was evaluated based on a disease index (DI), and nematicidal activity was assessed based on the total number of galls and nematode females per root gram. NPs successfully inhibited the growth of both pathogens without causing phytotoxicity on lettuce. The DI were significantly decreased as compared to the positive control (DI = 5.2), estimated equal to 1.7, 2.9 and 2.5 for Cu@PEG NPs, Cu-doped ZnO@DEG NPs and the chemical control (KOCIDE 2000), respectively. The reduction in galling and population of M. javanica ranged from 39.32% to 32.29%, statistically like chemical control. The treatment of lettuce plants with Cu-doped ZnO@DEG NPs increased the leaf net photosynthetic value at 4.60 and 6.66 µmol CO2-2 s-1 in plants inoculated with S. sclerotiorum and M. javanica, respectively, as compared to the control (3.00 µmol CO2-2 s-1). The antioxidant capacity of NPs treated lettuce plants was evaluated as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity in leaf extracts. Plants inoculated with S. sclerotiorum and sprayed with Cu-doped ZnO@DEG and Cu@PEG NPs, exhibited a 34.22% and 32.70% increase in antioxidant capacity, respectively, higher than the control. Similarly, an increase in antioxidant capacity was measured (39.49 and 37.36%) in lettuce inoculated with M. javanica and treated with Cu-doped ZnO@DEG and Cu@PEG NPs, respectively. Moreover, an increase of phenolic compounds in lettuce leaf tissue treated with NPs was measured as compared to the control. Overall, foliar applied Cu and Cu-doped ZnO NPs could be a promising tool to control phytopathogenic fungi and nematodes contributing to sustainability of agri-food sector.

Impact of Geraniol and Geraniol Nanoemulsions on Botrytis cinerea and Effect of Geraniol on Cucumber Plants' Metabolic Profile Analyzed by LC-QTOF-MS.

In the present study, the bioactive substance geraniol was tested in vitro and in planta against B. cinerea on cucumber plants, and the changes in the metabolic profile of cucumber plants inoculated with the pathogen and/or treated with geraniol were monitored by a novel LC-QTOF-MS method employing target and suspect screening. The aforementioned treatments were also studied for their impact on membrane lipid peroxidation calculated as malondialdehyde (MDA) content. Additionally, geraniol-loaded nanoemulsions (GNEs) were synthesized and tested against B. cinerea as an integrated formulation mode of geraniol application. The EC50 values calculated for geraniol and GNEs against B. cinerea were calculated at 235 µg/mL and 105 µg/mL, respectively. The in planta experiment on cucumber plants demonstrated the ability of geraniol and GNEs to significantly inhibit B. cinerea under greenhouse conditions. The LC-QTOF-MS analysis of the metabolic profile of the cucumber plants treated with geraniol demonstrated an increase in the concentration levels of myricetin, chlorogenic acid, and kaempferol rhamnoside, as compared to control plants and the presence of B. cinerea caused an increase in sinapic acid and genistein. These compounds are part of important biosynthetic pathways mostly related to responses against a pathogen attack.

An Optimized HPLC-DAD Methodology for the Determination of Anthocyanins in Grape Skins of Red Greek Winegrape Cultivars (Vitis vinifera L.).

A rapid and simple HPLC-DAD analytical method was developed and optimized for the determination of anthocynanins in three red Greek winegrape varieties (Kotsifali, Limnio, and Vradiano). The critical parameters, such as the acidifying solvent and the extraction temperature, which affect the extraction of anthocyanins from the grapes, were studied to find the optimum values. The developed methodology was validated in terms of selectivity, linearity, accuracy, and precision and presented satisfactory results. The limits of quantification (LOQs) ranged between 0.20 mg/kg to 0.60 mg/kg, and the limits of detection (LODs) ranged between 0.06 mg/kg and 0.12 mg/kg. The RSD% of the within-day and between-day assays were lower than 6.2% and 8.5%, respectively, showing adequate precision. The accuracy ranged between 91.6 and 119% for within-day assay and between 89.9 and 123% for between-day assay. Sixteen samples from the main regions of each variety as well as from the official ampelographic collections of Greece were collected during the 2020 growing season and were further analyzed by HPLC-DAD. Notable differences in the anthocyanin content were detected among the cultivars using hierarchical cluster analysis (HCA).

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota.

Dicyandiamide (DCD) and nitrapyrin (NP) are nitrification inhibitors (NIs) used in agriculture for over 40 years. Recently, ethoxyquin (EQ) was proposed as a novel potential NI, acting through its derivative quinone imine (QI). Still, the specific activity of these NIs on the different groups of ammonia-oxidizing microorganisms (AOM), and mostly their effects on other soil microbiota remain unknown. We determined the impact of QI, and comparatively of DCD and NP, applied at two doses (regular versus high), on the function, diversity, and dynamics of target (AOM), functionally associated (nitrite-oxidizing bacteria-NOB), and off-target prokaryotic and fungal communities in two soils mainly differing in pH (5.4 versus 7.9). QI was equally effective to DCD but more effective than NP in inhibiting nitrification in the acidic soil, while in the alkaline soil QI was less efficient than DCD and NP. This was attributed to the higher activity of QI toward AOA prevailing in the acidic soil. All NIs induced significant effects on the composition of the AOB community in both soils, unlike AOA, which were less responsive. Beyond on-target effects, we noted an inhibitory effect of all NIs on the abundance of NOB in the alkaline soil, with Nitrobacter being more sensitive than Nitrospira. QI, unlike the other NIs, induced significant changes in the composition of the bacterial and fungal communities in both soils. Our findings have serious implications for the efficiency and future use of NIs on agriculture and provide unprecedented evidence for the potential off-target effects of NIs on soil microbiota. IMPORTANCE NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms. Here, we studied the behavior of a new potent NI, QI, compared to established NIs. We show that (i) the variable efficacy of NIs across soils with different pH reflects differences in the inherent specific activity of the NIs to AOA and AOB; (ii) beyond AOM, NIs exhibit negative effects on other nitrifiers, like NOB; (iii) QI was the sole NI that significantly affected prokaryotic and fungal diversity. Our findings (i) highlight the need for novel NI strategies that consider the variable sensitivity of AOM groups to the different NIs (ii) identify QI as a potent AOA inhibitor, and (iii) stress the need for monitoring NIs' impact on off-target soil microorganisms to ensure sustainable N fertilizers use and soil ecosystem functioning.

Root Transcriptional and Metabolic Dynamics Induced by the Plant Growth Promoting Rhizobacterium (PGPR) Bacillus subtilis Mbi600 on Cucumber Plants.

Bacillus subtilis MBI600 is a commercialized plant growth-promoting bacterial species used as a biocontrol agent in many crops, controlling various plant pathogens via direct or indirect mechanisms. In the present study, a detailed transcriptomic analysis of cucumber roots upon response to the Bs MBI600 strain is provided. Differentially expressed genes (DEGs) analysis showed altered gene expression in more than 1000 genes at 24 and 48 h post-application of Bs MBI600. Bs MBI600 induces genes involved in ISR and SAR signaling. In addition, genes involved in phytohormone production and nutrient availability showed an upregulation pattern, justifying the plant growth promotion. Biocontrol ability of Bs MBI600 seems also to be related to the activation of defense-related genes, such as peroxidase, endo-1,3(4)-beta-glucanase, PR-4, and thaumatin-like. Moreover, KEGG enriched results showed that differentially expressed genes were classified into biocontrol-related pathways. To further investigate the plant's response to the presence of PGPR, a profile of polar metabolites of cucumber treated with Bs MBI600 was performed and compared to that of untreated plants. The results of the current study gave insights into the mechanisms deployed by this biocontrol agent to promote plant resistance, helping to understand the molecular interactions in this system.

CuZn and ZnO Nanoflowers as Nano-Fungicides against Botrytis cinerea and Sclerotinia sclerotiorum: Phytoprotection, Translocation, and Impact after Foliar Application.

Inorganic nanoparticles (INPs) have dynamically emerged in plant protection. The uptake of INPs by plants mostly depends on the size, chemical composition, morphology, and the type of coating on their surface. Herein, hybrid ensembles of glycol-coated bimetallic CuZn and ZnO nanoparticles (NPs) have been solvothermally synthesized in the presence of DEG and PEG, physicochemically characterized, and tested as nano-fungicides. Particularly, nanoflowers (NFs) of CuZn@DEG and ZnO@PEG have been isolated with crystallite sizes 40 and 15 nm, respectively. Organic coating DEG and PEG (23% and 63%, respectively) was found to protect the NFs formation effectively. The CuZn@DEG and ZnO@PEG NFs revealed a growth inhibition of phytopathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum in a dose-dependent manner with CuZn@DEG NFs being more efficient against both fungi with EC50 values of 418 and 311 µg/mL respectively. Lettuce (Lactuca sativa) plants inoculated with S. sclerotiorum were treated with the NFs, and their antifungal effect was evaluated based on a disease index. Plants sprayed with ZnO@PEG NFs showed a relatively higher net photosynthetic (4.70 µmol CO2 m-2s-1) and quantum yield rate (0.72) than with CuZn@DEG NFs (3.00 µmol CO2 m-2s-1 and 0.68). Furthermore, the penetration of Alizarin Red S-labeled NFs in plants was investigated. The translocation from leaves to roots through the stem was evident, while ZnO@PEG NFs were mainly trapped on the leaves. In all cases, no phytotoxicity was observed in the lettuce plants after treatment with the NFs.

Impacts of Decaying Aromatic Plants on the Soil Microbial Community and on Tomato Seedling Growth and Metabolism: Suppression or Stimulation?

This study provides insight into changes in the features of tomato seedlings growing in soils enriched with spearmint, peppermint, or rosemary leaves and into changes in the microbial communities of these soils used as seedbeds; an organic amendment was also applied as a positive control. While the soil microbial community flourished in the presence of all three aromatic plants, tomato growth was inhibited or stimulated depending on the plant that was used. More specifically, phospholipid fatty acid (PLFA) analysis showed an increase in the total microbial biomass and in the biomass of all the groups examined, except for actinobacteria, and changes in the microbial community structure, with Gram-negative bacteria and fungi being favoured in the mint treatments, in which the microbial biomass was maximized. Seedlings from the rosemary treatment were entirely inhibited; they were at the open-cotyledon stage throughout the experiment. Seedlings from the mint treatments were the heaviest, longest, and had the highest chlorophyll content and photosynthetic yield. Metabolomic analysis showed metabolism enhancement associated with both growth and priming in seedlings from the mint treatments and disruption of metabolic pathways in those from the rosemary treatment. There is a great potential for applying these aromatic plants as soil amendments and as either biostimulants of plant growth or as herbicides.

Comparison of Novel and Established Nitrification Inhibitors Relevant to Agriculture on Soil Ammonia- and Nitrite-Oxidizing Isolates.

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agro-ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB (Nitrosomonas europaea, Nitrosospira multiformis), ammonia-oxidizing archaea (AOA; "Candidatus Nitrosocosmicus franklandus," "Candidatus Nitrosotalea sinensis"), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC50 = 0.3-0.7 µM) compared to EQ (EC50 = 1-1.4 µM) and EQNL (EC50 = 26.6-129.5 µM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC50 = 221.9-248.7 µM vs EC50 = 0.6-2.1 µM). NP was the only NI to which both AOA and AOB were equally sensitive with EC50s of 0.8-2.1 and 1.0-6.7 µM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO.

Thymus Citriodorus (Schreb) Botanical Products as Ecofriendly Nematicides with Bio-Fertilizing Properties.

In recent years, interest has surged in the development of plant extracts into botanical nematicides as ecofriendly plant protection products. Aromatic plants are maybe the most studied category of botanicals used in this direction and the yielding essential oils are obtained on a commodity scale by hydro distillation. Nevertheless, can the bioactivity of aromatic plants always be attributed to the terpenes content? What would it mean for soil microcosms to bear the treatment of an essential oil to cure against Meloidogyne sp.? Are there other extraction procedures to prepare more ecofriendly botanical products starting from an aromatic material? Lemon thyme is studied herein for the first time for its nematicidal potential. We compare the efficacy of lemon thyme powder, macerate, water extract and essential oil to control Meloidogyne incognita (Chitwood) and Meloidogyne javanica (Chitwood), and we additionally study the secondary effects on soil microbes and free-living nematodes, as well as on tomato plant growth. According to our results lemon thyme powder enhances tomato plants' growth in a dose-response manner and when it is incorporated in soil at 1 g kg-1, it exhibits nematicidal activity at a 95% level on M. incognita. The water extract yielding from the same dose is nematicidal only if it is left unfiltered; otherwise only a paralysis effect is demonstrated but inside the soil the biological cycle of the pest is not arrested. The essential oil is good both in performing paralysis and biological cycle arrest, but it detrimentally lowers abundances of bacterial and fungal feeding nematodes. On the contrary, lemon thyme powder and unfiltered water extract augments the bacterial biomass, while the latter also increases the bacterivorous nematodes. Overall, the bio fertilizing lemon thyme powder and its unfiltered water extract successfully control root knot nematodes and are beneficial to soil microbes and saprophytic nematodes.

Whey: The Soil Bio-Community Enhancer That Selectively Controls Root-Knot Nematodes.

To date, it is mandatory for ecofriendly pest-management tools to be used in agriculture. Whey is a dairy-processing waste, a plant and soil chemical and fungicidal basic substance. The beneficial effect of whey on soil microorganisms, enzymatic activities, and free-living nematodes-combined with its toxic activity on the plant parasites-forms root knot nematodes. In this study, this finding is reported for the first time. A drip-irrigating tomato plant combined with whey in water at 3.125% (v/w) and 6.25% (v/w) dose dependently promoted Gram+ and Gram- bacteria, actinomycetes, and fungi biomass. Respectively, whey treatment and duration augmented the bacterial feeding nematodes along with the soil enzymatic activities, e.g., alkaline phosphatase, dehydrogenase, and urease. The counterpart for these soil organisms' and enzymes' functionality is the decomposition of organic matter, nutrient mineralization and cycling. Additionally, whey applied at 6.25% (v/w) every 10 days in a field experiment exhibited an efficacy of 70% on root knot nematodes. It is calculated that the EC50/3d value paralyzes in vitro Meloidogyne javanica, which was 3.2% (v/v). Conclusively, the soil application of whey could be a sustainable and ecofriendly method to combat the root knot nematodes and additionally to enhance soil biotic components.

The effects of arbuscular mycorrhizal fungi and essential oil on soil microbial community and N-related enzymes during the fungal early colonization phase.

The arbuscular mycorrhizal fungi (AMF) and the essential oils are both agents of sustainable agriculture, and their independent effects on the community of free-living soil microbes have been explored. In a tomato pot experiment, conducted in a sandy loam mixture, we examined the independent and joint effects of inoculation with the fungus Rhizophagous irregularis and the addition of Mentha spicata essential oil on the structure of the soil microbial community and the activity of soil enzymes involved in the N-cycle, during the pre-symbiosis phase. Plants were grown for 60 days and were inoculated with R. irregularis. Then pots were treated with essential oil (OIL) weekly for a period of a month. Two experimental series were run. The first targeted to examine the effect of inoculation on the microbial community structure by the phospholipid fatty acids analysis (PLFAs), and enzyme activity, and the second to examine the effects of inoculation and essential oil addition on the same variables, under the hypothesis that the joint effect of the two agents would be synergistic, resulting in higher microbial biomass compared to values recorded in singly treated pots. In the AMF pots, N-degrading enzyme activity was dominated by the activity of urease while in the non-inoculated ones by the activities of arylamidase and glutaminase. Higher microbial biomass was found in singly-treated pots (137 and 174% higher in AMF and OIL pots, respectively) compared with pots subjected to both treatments. In these latter pots, higher activity of asparaginase (202 and 162% higher compared to AMF and OIL pots, respectively) and glutaminase (288 and 233% higher compared to AMF and OIL pots, respectively) was found compared to singly-treated ones. Soil microbial biomasses and enzyme activity were negatively associated across all treatments. Moreover, different community composition was detected in pots only inoculated and pots treated only with oil. We concluded that the two treatments produced diverging than synergistic effects on the microbial community composition whereas their joint effect on the activity of asparaginase and glutaminase were synergistic.

Acetic Acid, 2-Undecanone, and (E)-2-Decenal Ultrastructural Malformations on Meloidogyne incognita.

The use of natural compounds to control phytonematodes is significantly increasing, as most of the old synthetic pesticides have been banned due to their eco-hostile character. Plant secondary metabolites are now evaluated as biologically active molecules against Meloidogyne spp. but their target site in the nematode body is rarely specified. Herein, we report on the ultrastructure modifications of the Meloidogyne incognita J2 after treatment with nematicidal plant secondary metabolites, that is acetic acid, (E)-2-decenal, and 2-undecanone. The commercial nematicide fosthiazate acting on acetylcholinesterase was used as control. For this reason, scanning electron microscopy and transmission electron microscopy have been employed. The acetic acid mainly harmed the cuticle, degenerated the nuclei of pseudocoel cells, and vacuolised the cytoplasm. The (E)-2-decenal and 2-undecanone did neither harm to the cuticle nor the somatic muscles but they degenerated the pseudocoel cells. (E)-2-decenal caused malformation of somatic muscles. According to the above, the nematicidal compounds seem to enter the nematode body principally via the digestive system rather than the cuticle, since the main part of the damage is internal.

Land Spreading of Wastewaters from the Fruit-Packaging Industry and Potential Effects on Soil Microbes: Effects of the Antioxidant Ethoxyquin and Its Metabolites on Ammonia Oxidizers.

Thiabendazole (TBZ), imazalil (IMZ), ortho-phenylphenol (OPP), diphenylamine (DPA), and ethoxyquin (EQ) are used in fruit-packaging plants (FPP) with the stipulation that wastewaters produced by their application would be depurated on site. However, no such treatment systems are currently in place, leading FPP to dispose of their effluents in agricultural land. We investigated the dissipation of those pesticides and their impact on soil microbes known to have a key role on ecosystem functioning. OPP and DPA showed limited persistence (50% dissipation time [DT50], 0.6 and 1.3 days) compared to TBZ and IMZ (DT50, 47.0 and 150.8 days). EQ was rapidly transformed to the short-lived quinone imine (QI) (major metabolite) and the more persistent 2,4-dimethyl-6-ethoxyquinoline (EQNL) (minor metabolite). EQ and OPP exerted significant inhibition of potential nitrification, with the effect of the former being more persistent. This was not reflected in the abundance (determined by quantitative PCR [qPCR]) of the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Considering the above discrepancy and the metabolic pattern of EQ, we further investigated the hypothesis that its metabolites and not only EQ were toxic to ammonia oxidizers. Potential nitrification, amoA gene abundance, and amoA gene transcripts of AOB and AOA showed that QI was probably responsible for the inhibition of nitrification. Our findings have serious ecological and practical implications for soil productivity and N conservation in agriculturally impacted ecosystems and stress the need to include metabolites and RNA-based methods when the soil microbial toxicity of pesticides is assessed.

The potential of organic substrates based on mushroom substrate and straw to dissipate fungicides contained in effluents from the fruit-packaging industry - Is there a role for Pleurotus ostreatus?

Citrus fruit-packaging plants (FPP) produce large wastewater volumes with high loads of fungicides like ortho-phenylphenol (OPP) and imazalil (IMZ). No methods are in place for the treatment of those effluents and biobeds appear as a viable alternative. We employed a column study to investigate the potential of spent mushroom substrate (SMS) of Pleurotus ostreatus, either alone or in mixture with straw and soil plus a mixture of straw /soil to retain and dissipate IMZ and OPP. The role of P. ostreatus on fungicides dissipation was also investigated by studying in parallel the performance of fresh mushroom substrate of P. ostreatus (FMS) and measuring lignolytic enzymatic activity in the leachates. All substrates effectively reduced the leaching of OPP and IMZ which corresponded to 0.014-1.1% and 0.120-0.420% of their initial amounts respectively. Mass balance analysis revealed that FMS and SMS/Straw/Soil (50/25/25 by vol) offered the most efficient removal of OPP and IMZ from wastewaters respectively. Regardless of the substrate, OPP was restricted in the top 0-20cm of the columns and was bioavailable (extractable with water), compared to IMZ which was less bioavailable (extractable with acetonitrile) but diffused at deeper layers (20-50, 50-80cm) in the SMS- and Straw/Soil-columns. PLFAs showed that fungal abundance was significantly lower in the top layer of all substrates from where the highest pesticide amounts were recovered suggesting an inhibitory effect of fungicides on total fungi in the substrates tested. Our data suggest that biobeds packed with SMS-rich substrates could ensure the efficient removal of IMZ and OPP from wastewaters of citrus FPP.

Nematicidal Activity of the Volatilome of Eruca sativa on Meloidogyne incognita.

Research on new pesticides based on plant extracts, aimed at the development of nontoxic formulates, has recently gained increased interest. This study investigated the use of the volatilome of rucola (Eruca sativa) as a powerful natural nematicidal agent against the root-knot nematode, Meloidogyne incognita. Analysis of the composition of the volatilome, using GC-MS-SPME, showed that the compound (Z)-3-hexenyl acetate was the most abundant, followed by (Z)-3-hexen-1-ol and erucin, with relative percentages of 22.7 ± 1.6, 15.9 ± 2.3, and 8.6 ± 1.3, respectively. Testing of the nematicidal activity of rucola volatile compounds revealed that erucin, pentyl isothiocyanate, hexyl isothiocyanate, (E)-2-hexenal, 2-ethylfuran, and methyl thiocyanate were the most active with EC50 values of 3.2 ± 1.7, 11.1 ± 5.0, 11.3 ± 2.6, 15.0 ± 3.3, 16.0 ± 5.0, and 18.1 ± 0.6 mg/L, respectively, after 24 h of incubation. Moreover, the nematicidal activity of fresh rucola used as soil amendant in a containerized culture of tomato decreased the nematode infection in a dose-response manner (EC50 = 20.03 mg/g) and plant growth was improved. On the basis of these results, E. sativa can be considered as a promising companion plant in intercropping strategies for tomato growers to control root-knot nematodes.

Nematicidal activity of furanocoumarins from parsley against Meloidogyne spp.

BACKGROUND: This report describes activity against Meloidogyne spp. and chemical characterisation of the essential oil and methanol extract of Petroselinum crispum aerial parts. The study was based on the hypothesis that P. crispum could be used as an intercrop and soil amendment in tomato culture for nematode control. RESULTS: The methanol extract and the essential oil exhibited significant nematicidal activity against M. incognita, M. hapla and M. arenaria, the first being the most sensitive species, with EC50 /72 h values of 140 ± 15 and 795 ± 125 mg L(-1) for the extract and oil respectively. The most abundant furanocoumarin compounds in the methanolic extract were xanthotoxin, psoralen, bergapten and oxypeucedanin; levels ranged from 1.77 to 46.04 mg kg(-1) wet weight. The EC50 /24 h values of xanthotoxol, psoralen and xanthotoxin against M. incognita were 68 ± 33, 147 ± 88 and 200 ± 21 mg L(-1) respectively. The addition of fresh parsley paste to soil reduced the number of M. incognita females and plant galls on tomato roots; EC50 values were 24.79 and 28.07 mg g(-1) respectively. Moreover, parsley paste enhanced tomato growth in a dose-response manner. CONCLUSIONS: Parsley exhibits promising nematicidal activity as an organic amendment and as a source of nematotoxic furanocoumarins.