Michalis Omirou

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.

Exploring the potential of biobeds for the depuration of pesticide-contaminated wastewaters from the citrus production chain: laboratory, column and field studies.

The high wastewater volumes produced during citrus production at pre- and post-harvest level presents serious pesticide point-source pollution for groundwater bodies. Biobeds are used for preventing such point-source pollution occurring at farm level. We explored the potential of biobeds for the depuration of wastewaters produced through the citrus production chain following a lab-to-field experimentation. The dissipation of pesticides used pre- or post-harvest was studied in compost-based biomixtures, soil, and a straw-soil mixture. A biomixture of composted grape seeds and skins (GSS-1) showed the highest dissipation capacity. In subsequent column studies, GSS-1 restricted pesticides leaching even at the highest water load (462 Lm(-3)). Ortho-phenylphenol was the most mobile compound. Studies in an on-farm biobed filled with GSS-1 showed that pesticides were fully retained and partially or fully dissipated. Overall biobeds could be a valuable solution for the depuration of wastewaters produced at pre- and post-harvest level by citrus fruit industries.

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.

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.

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.

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-, β- 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.