Above- and below-ground microbiome in the annual developmental cycle of two olive tree varieties.

The olive tree is a hallmark crop in the Mediterranean region. Its cultivation is characterized by an enormous variability in existing genotypes and geographical areas. As regards the associated microbial communities of the olive tree, despite progress, we still lack comprehensive knowledge in the description of these key determinants of plant health and productivity. Here, we determined the prokaryotic, fungal and arbuscular mycorrhizal fungal (AMF) microbiome in below- (rhizospheric soil, roots) and above-ground (phyllosphere and carposphere) plant compartments of two olive varieties 'Koroneiki' and 'Chondrolia Chalkidikis' grown in Southern and Northern Greece respectively, in five developmental stages along a full fruit-bearing season. Distinct microbial communities were supported in above- and below-ground plant parts; while the former tended to be similar between the two varieties/locations, the latter were location specific. In both varieties/locations, a seasonally stable root microbiome was observed over time; in contrast the plant microbiome in the other compartments were prone to changes over time, which may be related to seasonal environmental change and/or to plant developmental stage. We noted that olive roots exhibited an AMF-specific filtering effect (not observed for bacteria and general fungi) onto the rhizosphere AMF communities of the two olive varieties/locations/, leading to the assemblage of homogenous intraradical AMF communities. Finally, shared microbiome members between the two olive varieties/locations include bacterial and fungal taxa with putative functional attributes that may contribute to olive tree tolerance to abiotic and biotic stress.

Interplay between rhizobial nodulation and arbuscular mycorrhizal fungal colonization in Lotus japonicus roots.

AIMS: This study aims to identify main factors that influence the tripartite association of legumes with arbuscular mycorrhiza fungi (AMF) and nitrogen-fixing rhizobia. METHODS AND RESULTS: Concurrent inoculations with Mesorhizobium loti and four AMF strains were performed on the model legume Lotus japonicus. Nodulation was significantly enhanced by all AMF strains, under normal conditions, and by specific AMF strains under heat-stress conditions. The impact of rhizobia on mycorrhizal colonization was AMF strain dependent. Co-inoculation trials, where either AMF or rhizobia were restricted outside the root, showed that the symbiotic phenotypes are not influenced by microbial interactions at the pre-symbiotic stage. External application of nutrients showed that P enhances nodulation, while N application does not enhance mycorrhizal colonization. CONCLUSIONS: Nodulation and mycorhization affect one another during advanced stages of the symbiosis. AMF strains may enhance nodulation under both normal and high environmental temperatures. Rhizobium-AMF compatibility is critical, as rhizobium may positively affect specific AMF strains, an effect that does not derive from increased N uptake.

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.

Multi-species relationships in legume roots: From pairwise legume-symbiont interactions to the plant - microbiome - soil continuum.

Mutualistic relationships of legume plants with, either bacteria (like rhizobia) or fungi (like arbuscular mycorrhizal fungi), have been investigated intensively, usually as bi-partite interactions. However, diverse symbiotic interactions take place simultaneously or sequentially under field conditions. Their collective, but not additive, contribution to plant growth and performance remains hard to predict, and appears to be furthermore affected by crop species and genotype, non-symbiotic microbial interactions and environmental variables. The challenge is: (i) to unravel the complex overlapping mechanisms that operate between the microbial symbionts as well as between them, their hosts and the rhizosphere (ii) to understand the dynamics of the respective mechanisms in evolutionary and ecological terms. The target for agriculture, food security and the environment, is to use this insight as a solid basis for developing new integrated technologies, practices and strategies for the efficient use of beneficial microbes in legumes and other plants. We review recent advances in our understanding of the symbiotic interactions in legumes roots brought about with the aid of molecular and bioinformatics tools. We go through single symbiont-host interactions, proceed to tripartite symbiont-host interactions, appraise interactions of symbiotic and associative microbiomes with plants in the root-rhizoplane-soil continuum of habitats and end up by examining attempts to validate community ecology principles in the legume-microbe-soil biosystem.

Integrated biodepuration of pesticide-contaminated wastewaters from the fruit-packaging industry using biobeds: Bioaugmentation, risk assessment and optimized management.

Wastewaters from fruit-packaging plants contain high loads of toxic and persistent pesticides and should be treated on site. We evaluated the depuration performance of five pilot biobeds against those effluents. In addition we tested bioaugmentation with bacterial inocula as a strategy for optimization of their depuration capacity. Finally we determined the composition and functional dynamics of the microbial community via q-PCR. Practical issues were also addressed including the risk associated with the direct environmental disposal of biobed-treated effluents and decontamination methods for the spent packing material. Biobeds showed high depuration capacity (>99.5%) against all pesticides with bioaugmentation maximizing their depuration performance against the persistent fungicide thiabendazole (TBZ). This was followed by a significant increase in the abundance of bacteria, fungi and of catabolic genes of aromatic compounds catA and pcaH. Bioaugmentation was the most potent decontamination method for spent packing material with composting being an effective alternative. Risk assessment based on practical scenarios (pome and citrus fruit-packaging plants) and the depuration performance of the pilot biobeds showed that discharge of the treated effluents into an 0.1-ha disposal site did not entail an environmental risk, except for TBZ-containing effluents where a larger disposal area (0.2ha) or bioaugmentation alleviated the risk.

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.

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.

Olive mill wastewater biodegradation potential of white-rot fungi--Mode of action of fungal culture extracts and effects of ligninolytic enzymes.

Forty-nine white-rot strains belonging to 38 species of Basidiomycota were evaluated for olive-mill wastewater (OMW) degradation. Almost all fungi caused high total phenolics (>60%) and color (⩽ 70%) reduction, while COD and phytotoxicity decreased to a lesser extent. Culture extracts from selected Agrocybe cylindracea, Inonotus andersonii, Pleurotus ostreatus and Trametes versicolor strains showed non-altered physicochemical and enzymatic activity profiles when applied to raw OMW in the presence or absence of commercial catalase, indicating no interaction of the latter with fungal enzymes and no competition for H2O2. Hydrogen peroxide's addition resulted in drastic OMW's decolorization, with no effect on phenolic content, suggesting that oxidation affects colored components, but not necessarily phenolics. When fungal extracts were heat-treated, no phenolics decrease was observed demonstrating thus their enzymatic rather than physicochemical oxidation. Laccases added to OMW were reversibly inhibited by the effluent's high phenolic load, while peroxidases were stable and active during the entire process.

Use of residues and by-products of the olive-oil production chain for the removal of pollutants from environmental media: A review of batch biosorption approaches.

Residues and by-products of the olive-oil production chain have been widely studied as biosorbents for the removal of various pollutants from environmental media due to their significant adsorption properties, low cost, production at local level and renewability. In this review, adsorbents developed from olive-tree cultivation residues and olive-oil extraction by-products and wastes are examined, and their sorption characteristics are described and discussed. Recent information obtained using batch sorption studies is summarized and the adsorption mechanisms involved, regarding various aquatic and soil pollutants (metal ions, dyes, radionuclides, phenolic compounds, pesticides) are presented and discussed. It is evident that several biosorbents show the potential to effectively remove a wide variety of pollutants from aqueous solutions, especially Pb and Cd. However, there is need to (a) develop standardized batch study protocols, and potentially reference materials, for effective cross-evaluation of biosorbents of similar nature and for improved understanding of mechanisms involved and (b) investigate scaling-up and regeneration issues that hold back industry-level application of preselected adsorbents.

Biodegradation and detoxification of olive mill wastewater by selected strains of the mushroom genera Ganoderma and Pleurotus.

Thirty-nine white-rot fungi belonging to nine species of Agaricomycotina (Basidiomycota) were initially screened for their ability to decrease olive-mill wastewater (OMW) phenolics. Four strains of Ganoderma australe, Ganoderma carnosum, Pleurotus eryngii and Pleurotus ostreatus, were selected and further examined for key-aspects of the OMW biodegradation process. Fungal growth in OMW-containing batch cultures resulted in significant decolorization (by 40-46% and 60-65% for Ganoderma and Pleurotus spp. respectively) and reduction of phenolics (by 64-67% and 74-81% for Ganoderma and Pleurotus spp. respectively). COD decrease was less pronounced (12-29%). Cress-seeds germination increased by 30-40% when OMW was treated by Pleurotus strains. Toxicity expressed as inhibition of Aliivibrio fischeri luminescence was reduced in fungal-treated OMW samples by approximately 5-15 times compared to the control. As regards the pertinent enzyme activities, laccase and Mn-independent peroxidase were detected for Ganoderma spp. during the entire incubation period. In contrast, Pleurotus spp. did not exhibit any enzyme activities at early growth stages; instead, high laccase (five times greater than those of Ganoderma spp.) and Mn peroxidases activities were determined at the end of treatment. OMW decolorization by Ganoderma strains was strongly correlated to the reduction of phenolics, whereas P. eryngii laccase activity was correlated with the effluent's decolorization.

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.

Potential for bioremediation of agro-industrial effluents with high loads of pesticides by selected fungi.

Wastewaters from the fruit packaging industry contain a high pesticide load and require treatment before their environmental discharge. We provide first evidence for the potential bioremediation of these wastewaters. Three white rot fungi (WRF) (Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus) and an Aspergillus niger strain were tested in straw extract medium (StEM) and soil extract medium (SEM) for degrading the pesticides thiabendazole (TBZ), imazalil (IMZ), thiophanate methyl (TM), ortho-phenylphenol (OPP), diphenylamine (DPA) and chlorpyrifos (CHL). Peroxidase (LiP, MnP) and laccase (Lac) activity was also determined to investigate their involvement in pesticide degradation. T. versicolor and P. ostreatus were the most efficient degraders and degraded all pesticides (10 mg l⁻¹) except TBZ, with maximum efficiency in StEM. The phenolic pesticides OPP and DPA were rapidly degraded by these two fungi with a concurrent increase in MnP and Lac activity. In contrast, these enzymes were not associated with the degradation of CHL, IMZ and TM implying the involvement of other enzymes. T. versicolor degraded spillage-level pesticide concentrations (50 mg l⁻¹) either fully (DPA, OPP) or partially (TBZ, IMZ). The fungus was also able to rapidly degrade a mixture of TM/DPA (50 mg l⁻¹), whereas it failed to degrade IMZ and TBZ when supplied in a mixture with OPP. Overall, T. versicolor and P. ostreatus showed great potential for the bioremediation of wastewaters from the fruit packaging industry. However, degradation of TBZ should be also achieved before further scaling up.

Degradation and adsorption of pesticides in compost-based biomixtures as potential substrates for biobeds in southern Europe.

Biobeds have been used in northern Europe for minimizing point source contamination of water resources by pesticides. However, little is known regarding their use in southern Europe where edaphoclimatic conditions and agriculture practices significantly differ. A first step toward their adaptation in southern Europe is the use of low-cost and easily available substrates as biomixture components. This study investigated the possibility of replacing peat with agricultural composts in the biomixture. Five composts from local substrates including olive leaves, cotton crop residues, cotton seeds, spent mushroom substrate, and commercial sea wrack were mixed with topsoil and straw (1:1:2). Degradation of a mixture of pesticides (dimethoate, indoxacarb, buprofezin, terbuthylazine, metribuzin, metalaxyl-M, iprodione, azoxystrobin) at two dose rates was tested in the compost biomixtures (BX), in corresponding peat biomixtures (OBX), and in soil. Adsorption-desorption of selected pesticides were also studied. Pesticide residues were determined by gas chromatography with nitrogen-phosphorus detector, except indoxacarb, which was determined with a microelectron capture detector. Overall, BX degraded the studied pesticides at rates markedly higher than those observed in soil and OBX, in which the slowest degradation rates were evident. Overall, the olive leaf compost biomixture showed the highest degradation capacity. Adsorption studies showed that OBX and BX had higher adsorption affinity compared to soil. Desorption experiments revealed that pesticide adsorption in biomixtures was not entirely reversible. The results suggest that substitution of peat with local composts will lead to optimization of the biobed system for use in Mediterranean countries.

Characterization of cultivated fungi isolated from grape marc wastes through the use of amplified rDNA restriction analysis and sequencing.

Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7 x 10(6) per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.

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.

Effect of continuous olive mill wastewater applications, in the presence and absence of nitrogen fertilization, on the structure of rhizosphere-soil fungal communities.

Olive mill wastewater (OMW) is rich in potentially toxic organics precluding its disposal into water receptors. However, land application of diluted OMW may result in safe disposal and fertilization. In order to investigate the effects of OMW on the structure of soil fungal groups, OMW was applied daily to pepper plants growing in a loamy sand and a sandy loam at two doses for a period of 3 months (total OMW equivalents 900 and 1800 m(3) ha(-1)). Nitrogen (N) fertilization alleviated N scarcity and considerably enhanced plant biomass production; however, when applied in combination with the high OMW dose, it induced plant stress. OMW applications resulted in marked changes in the denaturing gradient gel electrophoresis patterns of soil basidiomycete communities, while concurrent N fertilization reduced these effects. In contrast, the ascomycete communities required N fertilization to respond to OMW addition. Cloning libraries for the basidiomycete communities showed that Cryptococcus yeasts and Ceratobasidium spp. dominated in the samples treated with OMW. In contrast, certain plant pathogenic basidiomycetes such as Thanatephorus cucumeris and Athelia rolfsii were suppressed. The observed changes may be reasonably explained by the capacity of OMW to enrich soils in organic substrates, to induce N immobilization and to directly introduce OMW-derived basidiomycetous yeasts.

Role of ethylene in the protection of tomato plants against soil-borne fungal pathogens conferred by an endophytic Fusarium solani strain.

An endophytic fungal isolate (Fs-K), identified as a Fusarium solani strain, was obtained from root tissues of tomato plants grown on a compost which suppressed soil and foliar pathogens. Strain Fs-K was able to colonize root tissues and subsequently protect plants against the root pathogen Fusarium oxysporum f.sp. radicis-lycopersici (FORL), and elicit induced systemic resistance against the tomato foliar pathogen Septoria lycopersici. Interestingly, attenuated expression of certain pathogenesis-related genes, i.e. PR5 and PR7, was detected in tomato roots inoculated with strain Fs-K compared with non-inoculated plants. The expression pattern of PR genes was either not affected or aberrant in leaves. A genetic approach, using mutant tomato plant lines, was used to determine the role of ethylene and jasmonic acid in the plant's response to infection by the soil-borne pathogen F. oxysporum f.sp. radicis-lycopersici (FORL), in the presence or absence of isolate Fs-K. Mutant tomato lines Never ripe (Nr) and epinastic (epi1), both impaired in ethylene-mediated plant responses, inoculated with FORL are not protected by isolate Fs-K, indicating that the ethylene signalling pathway is required for the mode of action used by the endophyte to confer resistance. On the contrary, def1 mutants, affected in jasmonate biosynthesis, show reduced susceptibility to FORL, in the presence Fs-K, which suggests that jasmonic acid is not essential for the mediation of biocontrol activity of isolate Fs-K.

Cytological and other aspects of pathogenesis-related gene expression in tomato plants grown on a suppressive compost.

BACKGROUND AND AIMS: Recent studies have shown that certain composts may trigger indirect defence mechanisms by sensitizing the plant to create an increased state of resistance, similar to systemic acquired resistance. In this study, the capacity of a disease-suppressive compost to alter the expression pattern of certain pathogenesis-related (PR) genes in the root system of tomato plants (Solanum lycopersicum) provided the opportunity to study their cellular expression pattern and to investigate putative roles of these genes in the mechanisms of plant defence. METHODS: Employing the reverse transcription-polymerase chain reaction (RT-PCR) and in situ RNA:RNA hybridization techniques, the accumulation and distribution of the transcripts of the differentially expressed PR genes were examined in plants grown on compost and compared with those of control plants grown on peat. KEY RESULTS: Elevated levels of expression of the pathogenesis-related genes PR-1, PR-5 and P69/PR-7 were detected in the roots of tomato plants grown on the compost. A clearly distinguished spatial induction pattern was observed for these PR genes: PR-1 transcripts were almost exclusively detected in the pericycle cells surrounding the root stele of the main and lateral roots; PR-5 transcripts were present in the phloem of the root and stem tissues; and the accumulation and distribution of PR-7 transcripts was detected in discrete groups of cells that appeared sporadically in both the parenchyma and vascular system of the root, suggesting that the gene is not expressed in a tissue-specific manner. In addition, a novel cDNA clone was isolated (P69G), which probably encodes a new tomato P69 isoform. CONCLUSIONS: This study provides evidence that a suppressive compost is able to elicit consistent and increased expression of certain PR genes in the roots of tomato plants, even in the absence of any pathogen. The in situ localization studies reveal expression patterns which are in accordance with the presence of protein or with the putative roles of the respective encoded proteins. The expression of the PR genes may be triggered by the microflora of the compost or could be associated with abiotic factors of the compost.

Ecophysiology and molecular phylogeny of bacteria isolated from alkaline two-phase olive mill wastes.

The use of two-phase centrifugal decanters has been widely adopted in the olive oil extraction industry in order to reduce the huge quantities of wastewaters produced during the traditional three-phase extraction process. The resulting sludge-like byproduct, widely known as "alpeorujo", has a pH of 4-6, low water activity (a(w)) and high phytotoxicity. Addition of Ca(OH)(2) to alpeorujo, which is commonly performed at the olive oil mill to handle disposal problems related to acidic pH and odor emissions, creates an alkaline secondary waste (alkaline alpeorujo). Bacteria isolated from alkaline alpeorujo were cultured in order to investigate their physiological and phylogenetic characteristics. The bacterial population at neutral pH was estimated to be 6.0+/-0.4 x 10(7) cells g(-1) dw, while the bacterial population at pH 11 reached 2.1+/-0.3 x 10(5) cells g(-1) dw. Fourteen strains isolated from alkaline pH were halotolerant alkaliphiles, while seven isolates from neutral pH were moderate to extreme halotolerant or/and alkalitolerant bacteria. Based on 16S rRNA gene sequence analysis, four of the halotolerant alkaliphilic isolates showed 98.4-99.2% similarity to known sequences of Bacillus alcalophilus and Nesterenkonia lacusekhoensis, whereas ten isolates demonstrated low percentage similarities (94.4-96.9%) to the genera Idiomarina, Halomonas and Nesterenkonia. As concerns bacteria isolated from neutral pH, four isolates were associated with Corynebacterium, Novosphingobium, Serratia marcescens and Pseudomonas aeruginosa (98.3-99.9% similarities), while three isolates presented 96.5-97.2% sequence similarities to Rhodobacter, Pseudomonas and Ochrobactrum. At least six groups of isolates represent novel phylogenetic linkages among Bacteria.

Bacterial diversity in spent mushroom compost assessed by amplified rDNA restriction analysis and sequencing of cultivated isolates.

Spent mushroom compost (SMC) is the residual by-product of commercial Agaricus spp. cultivation, and it is mainly composed of a thermally treated cereal straw/animal manure mixture colonized by the fungal biomass. Research on the valorization of this material is mainly focusing on its use as soil conditioner and plant fertilizer. An investigation of the bacterial diversity in SMC was performed using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils. The bacterial population was estimated by the plate count method to a mean of 2.7 10(9) colony forming units (cfu) per g of dry weight, while the numbers of Gram-positive and Gram-negative bacteria were 1.9 10(9) and 4.9 10(8) cfu per g dw respectively as estimated by enumeration on semi-selective media. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera Bacillus, Paenibacillus, Exiguobacterium, Staphylococcus, Desemzia, Carnobacterium, Brevibacterium, Arthrobacter and Microbacterium of the bacterial divisions Firmicutes and Actinobacteria. Two bacterial groups have phylogenetic links with the genera Comamonas and Sphingobacterium, which belong to beta-Proteobacteria and Bacteroidetes respectively. Two potentially novel bacteria are reported, which are associated with the genera Bacillus and Microbacterium. Most of the bacteria identified are of environmental origin, while strains related to species usually isolated from insects, animal and clinical sources were also detected. It appears that bacterial diversity in SMC is greatly affected by the origin of the initial material, its thermal pasteurization treatment and the potential unintended colonization of the mushroom substrate during the cultivation process.