First Record of Neofusicoccum luteum and Colletotrichum gloeosporioides Causing Anthracnose and Stem End Rot on Avocado Fruits in Greece.

In May 2022, rot symptoms were observed 5 days after storage on fresh avocado fruits cv "Lamb Hass" harvested from a 3.4 ha organic orchard in Chania, Crete exhibiting 30% symptom incidence. Brownish-green sunken lesions and soft rot with dark brown lesions covering up to 50% of the mesocarp on fruits and blackish soft lesions on fruit stem ends were observed. To isolate the pathogens, fruits were surface sterilized using 1% NaOCl for 1 min, placed in 70% ethanol for 30 s and washed twice with sterile distilled water. Then, small pieces were excised from the fruit rot margins and transferred on PDA amended with 0.015% streptomycin-sulfate. Single-spore isolates were incubated on PDA for 10 days and subjected to morphological examination. Two distinct pathogenic fungal isolates were obtained, and their symptoms were re-examined on avocado fruits. The first isolate (A1) obtained from the fruit stem end, initially produced hyaline dense aerial mycelia, being gray and black on the upper and lower surface of the Petri dishes, respectively. The second isolate (A2) obtained from the main body of the fruit, formed round, grayish colonies, with orange conidial aggregates. Based on morphological characteristics (Phillips et al.,2013; Weir et al., 2012), isolates were preliminary identified as Neofusicoccum sp. (A1) and Colletotrichum sp. (A2). Isolates were molecularly identified by sequencing of the ITS-5.8S rRNA, translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2) genes. PCRs were conducted using primer pairs ITS4/ITS5, EF1-728F/EF1986R and Bt2a/Bt2b as well as ITS4/ITS5 and 5'-tef1/3'-tef1 and Bt2a/Bt2b for isolates A1 and A2, respectively (Carbone & Kohn, 1999; Glass & Donaldson, 1995; Rojas et al., 2010; Weir et al., 2012; White et al., 1990). The sequences were deposited into GenBank under the accession numbers OQ852465, OQ867962, OQ867965 for N. luteum and, OQ852466, OQ867963 and OQ867964 for C. gloeosporioides. Based on Multilocus sequence analysis (MLSA), a phylogenetic tree was constructed using concatenated sequences, following Kimura's two parameter model (1980), which confirmed their identity as N luteum and C. gloeosporioides strains. Mature avocado fruits (cv. Hass) were surface sterilized and dried. Consequently, incised fruits were inoculated with mycelial agar plugs (5 mm in diameter) cut from the edge of rapidly growing colonies of N. luteum and C. gloeosporioides strains. Fruits incubated in moist chambers and at 25°C for 5 days in the dark. Fruit bodies and stems were inoculated with the respective isolates and sterile agar plugs in the case of the control. Five fruits were used for each pathogenic trial per fungal isolate, which was repeated twice. After symptom occurrence, these pathogenic isolates were re-isolated successfully and molecularly identified, while exhibiting similar to original symptoms confirming Koch's postulates. While other reports exist on the presence of these pathogens in different countries worldwide, this is the first report of C. gloeosporioides and N. luteum as post-harvest pathogens of avocado, which is an economically important crop of Crete, in Greece (Akgül et al., 2016). This study provides the means for the accurate identification of these fungal pathogens causing avocado fruit rots and taking into consideration the available treatment options can contribute to establishing effective management strategies.

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.

Metal nanoparticles against fungicide resistance: alternatives or partners?

Chemical control suffers from the loss of available conventional active ingredients due to strict environmental safety regulations which, combined with the loss of fungicide efficacy due to resistance development, constitute major problems of contemporary crop protection. Metal-containing nanoparticles (MNPs) appear to have all the credentials to be next-generation, eco-compatible fungicide alternatives and a valuable anti-resistance management tool. Could the introduction of MNPs as nano-fungicides be the answer to both reducing the environmental footprint of xenobiotics and dealing with fungicide resistance? The potential of MNPs to be utilized as nano-fungicides, both as alternatives to conventional fungicides or/and as partners in combating fungicide resistance, is discussed in terms of effectiveness, potential antimicrobial mechanisms as well as synergy profiles with conventional fungicides. However, their "golden" potential to be used both as alternatives and partners of conventional fungicides to combat resistance and reduce environmental pollution is challenged by undesirable effects towards non-target organisms such as phytotoxicity, toxicity to humans and environmental ecotoxicity, constituting risks that should be considered before their commercial introduction as nano-pesticides at a large scale. © 2022 Society of Chemical Industry.

Fungicide resistance frequencies of Botrytis cinerea greenhouse isolates and molecular detection of a novel SDHI resistance mutation.

Sensitivity of B. cinerea to commonly used fungicides against Gray mold with emphasis to the newer quinone outside inhibitor (QoIs), and succinate dehydrogenase inhibitors (SDHIs) was assessed during a monitoring survey from vegetable greenhouses in four representative regions of Crete. 42% from a total of 168 isolates were simultaneously resistant to boscalid, fluopyram, pyraclostrobin and fenhexamid but not to fludioxonil making this phenylpyrrole fungicide an excellent anti-resistance antifungal agent. Isolates with double resistance to SDHIs and QoIs were found in very high frequencies indicating a selection towards double resistance due to the use of pyraclostrobin-boscalid mixtures. A number of sdhB resistance mutations (H272R, N230I and P225F/H) were found in isolates also carrying the G143A cytb resistance mutation in the above isolates. A novel sdhB point mutation (I274V) was identified for the first time in B. cinerea isolates collected from greenhouses with a fluopyram spray history with specific resistance to SDHIs. A PCR-RFLP diagnostic assay was developed for the detection of this mutation in the sdhB gene. Mutations P225F/H and I274V were found to be associated with fitness penalties in terms of mycelial growth, sporulation or pathogenicity. Results suggest that, in order to retain effective control of gray mold in Crete, appropriate anti-resistance strategies should be implemented taking into account the high double SDHI and QoI resistance frequencies. Additional studies for monitoring the already known and the new SDHI-resistance mutations, are necessary in order to hinder the further spread and establishment of single or double resistant isolates of B. cinerea detected in greenhouses in Crete.

Zinc nanoparticles: Mode of action and efficacy against boscalid-resistant Alternaria alternata isolates.

The antifungal potential of ZnO-NPs against Alternaria alternata isolates with reduced sensitivity to the succinate dehydrogenase inhibitor (SDHI) boscalid, resulting from target site modifications, was evaluated in vitro and in vivo. ZnO-NPs could effectively inhibit mycelial growth in a dose-dependent way in both boscalid (BOSC) sensitive (BOSC-S) and resistant (BOSC-R) isolates. The fungitoxic effect of ZnO-NPs against the pathogen was significantly enhanced when combined with boscalid compared to the individual treatments in all phenotype cases (BOSC-S/R) both in vitro and in vivo. Fungitoxic effect of ZnO-NPs could be, at least partly, attributed to zinc ion release as indicated by the positive correlation between sensitivities to the nanoparticles and their ionic counterpart ZnSO4 and the alleviation of the ZnO-NPs fungitoxic action in the presence of the strong chelating agent EDTA. The superior effectiveness of ZnO-NPs against A. alternata, compared to ZnSO4, could be due to nanoparticle properties interfering with cellular ion homeostasis mechanisms. The observed additive action of the oxidative phosphorylation-uncoupler fluazinam (FM) against all phenotypes indicates a possible role of ATP-dependent ion efflux mechanism in the mode of action of ZnO-NPs. A potential role of ROS production in the fungitoxic action of ZnO-NPs was evident by the additive/synergistic action of salicylhydroxamate (SHAM), which blocks the alternative oxidase antioxidant action. Mixture of ZnO-NPs and boscalid, resulting in a "capping" effect for the nanoparticles and significantly reducing their mean size, probably accounted for the synergistic effect of the mixture against both sensitive and resistant A. alternata isolates. Summarizing, results indicated that ZnO-NPs can be effectively used against A. alternata both alone or in combination with boscalid, providing an effective tool for combating SDHI-resistance and reducing the environmental fingerprint of synthetic fungicides.

Metal nanoparticles: Phytotoxicity on tomato and effect on symbiosis with the Fusarium solani FsK strain.

The effect of copper (Cu-NPs, CuO-NPs), silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles (NPs) on plant growth, physiological properties of tomato plants and their symbiotic relationships with the endophytic Fusarium solani FsK strain was investigated. Fungitoxicity tests revealed that the FsK strain was significantly more sensitive to Cu-NPs and ZnO-NPs than CuO-NPs and Ag-NPs both in terms of mycelial growth and spore germination. All NPs were more toxic to FsK compared to their bulk counterparts except for AgNO3, which was 8 to 9-fold more toxic than Ag-NPs. Apart from AgNO3, NPs and bulk counterparts did not affect the number of germinated tomato seeds even in higher concentrations, while root length was significantly reduced in a dose dependent way in most cases. Dry weight of tomato plants was also significantly reduced upon treatment with NPs and counterparts with most pronounced effects in the cases of AgNO3, Cu-NPs, ZnO-NPs, and ZnSO4. Root and shoot length of grown tomato plants was also affected by treatments while differences between NPs and bulk counterparts varied. A marked oxidative stress response was recorded in all cases of NPs/bulk counterparts as indicated by increased MDA and H2O2 levels of treated plants. Treated plants had significantly reduced chlorophyl-a and carotenoid levels compared to the untreated control. NPs and counterparts did not affect FsK colonization of roots indicating a possible shielding effect of tomato plants once the endophyte was established inside the roots. Vice versa, a possible alleviation of CuO-NPs, ZnO-NPs, and ZnSO4 toxicity was observed in the presence of FsK inside tomato roots in terms of plant dry weight. The results suggest that phytotoxicity of NPs in tomato treated plants should be considered before application and while both FsK and tomato are sensitive to NPs, their reciprocal benefits may extent to resistance towards these toxic agents.

Copper nanoparticles against benzimidazole-resistant Monilinia fructicola field isolates.

Nano-fungicides are expected to play an important role in future plant disease management. Their unique properties include a broad antimicrobial action, increased effectiveness in lower doses, slower a.i. release and/or enhanced drug delivery and an ability to control drug-resistant pathogens, which makes them appealing candidates for use as eco-friendly antifungal alternatives to counter fungicides resistance. Copper nanoparticles (Cu-NPs) could suppress mycelial growth in both sensitive (BENS) and resistant (BEN-R) Monilinia fructicola isolates harboring the E198A benzimidazole resistance mutation, more effectively than copper oxide NPs (CuO-NPs) and Cu(OH)2. A significant synergy of Cu-NPs with thiophanate methyl (TM) was observed against BEN-S isolates both in vitro and when applied on plum fruit suggesting enhanced availability or nanoparticle induced transformation of TM to carbendazim. ATP-dependent metabolism is probably involved in the mode of fungitoxic action of Cu-NPs as indicated by the synergy observed between Cu-NPs and the oxidative phosphorylation-uncoupler fluazinam (FM). Copper ion release contributed in the toxic action of Cu-NPs against M. fructicola, as indicated by synergism experiments with ethylenediaminetetraacetic acid (EDTA), although the lack of correlation between nano and bulk/ionic copper forms indicate an additional nano-property mediated mechanism of fungitoxic action. Results suggested that Cu-NPs can be effectively used in future plant disease management as eco-friendly antifungal alternatives to counter fungicides resistance and reduce the environmental footprint of synthetic fungicides.

Use of silver nanoparticles to counter fungicide-resistance in Monilinia fructicola.

The potential of Ag-NPs to suppress Monilia fructicola isolates and to broaden the effectiveness of fungicides to overcome resistance was tested in vitro and in vivo. Twenty-three M. fructicola isolates were subjected to fungitoxicity screening with a number of fungicides in vitro, which resulted in the detection of 18 isolates resistant to benzimidazoles (BEN-R) thiophanare methyl (TM) and carbendazim (CARB). DNA sequencing revealed the E198A resistance mutation in the ß-tubulin gene, target site of the benzimidazole fungicides in all resistant isolates. Ag-NPs effectively suppressed mycelial growth in both sensitive (BENS) and resistant isolates. The combination of Ag-NPs with TM led to a significantly enhanced fungitoxic effect compared to the individual treatments regardless resistant phenotype (BEN-R/S) both in vitro and when applied on apple fruit. The above observed additive/synergistic action is probably associated with an enhanced Ag-NPs activity/availability as indicated by the positive correlation between Ag-NPs and TM + Ag-NPs treatments. No correlation was found between AgNO3 and Ag-NPs suggesting that difference(s) exist in the fungitoxic mechanism of action between nanoparticles and their ionic counterparts. Synergy observed between Ag-NPs and the oxidative phosphorylation-uncoupler fluazinam (FM) against both resistance phenotypes indicates a possible role of energy (ATP) metabolism in the mode of action of Ag-NPs. Additionally, the role of released silver ions on the fungitoxic action of Ag-NPs against M. fructicola was found to be limited because the combination with NaCl revealed a synergistic rather than the antagonistic effect that would be expected from silver ion binding with chlorine ions. The results of this study suggested that Ag-NPs can be effectively used against M. fructicola and when used in combination with conventional fungicides they could provide the means for countering benzimidazole resistance and at the same time reduce the environmental impact of synthetic fungicides by reducing doses needed for the control of the pathogen.

Synergy between Cu-NPs and fungicides against Botrytis cinerea.

Combating drug-resistance is a daunting task, especially due to the shortage of available drug alternatives with multisite modes of action. In this study, the potential of copper nanoparticles (Cu-NPs) to suppress 15 Botrytis cinerea isolates, which are sensitive or resistant to fungicides, alone or in combination with conventional fungicides, was tested in vitro and in vivo. Sensitivity screening in vitro revealed two fungicide resistance phenotypes, resulting from target site mutations. DNA sequencing revealed three B. cinerea isolates highly resistant to benzimidazoles (BEN-R), thiophanare methyl (TM), and carbendazim, bearing the E198A resistance mutation in the ß-tubulin gene, and four isolates highly resistant to the QoI pyraclostrobin (PYR-R) with a G143A mutation in the cytb gene. Cu-NPs were equally effective against sensitive and resistant isolates. An additive/synergistic effect was observed between Cu-NPs and TM in the case of BEN-S isolates both in vitro and when applied in apple fruit. A positive correlation was observed between TM and TM + Cu-NPs treatments, suggesting that an increased TM availability in the target site could be related with the observed additive/synergistic action. No correlation between Cu(OH)2 and Cu-NPs sensitivity was found, indicating that different mechanisms govern the fungitoxic activity between nano and bulk counterparts. A synergistic profile was observed between Cu-NPs and fluazinam (FM) - an oxidative phosphorylation inhibitor - in all isolates regardless of resistance phenotype, suggesting that ATP metabolism could be involved in the mode of action of Cu-NPs. Furthermore, the observed cross sensitivity and antagonistic action between Cu-NPs and NaCl also provided evidence for copper ions contribution to the fungitoxic action of Cu-NPs. The results suggested that Cu-NPs in combination with conventional fungicides can provide the means for an environmentally safe, sustainable resistance management strategy by reducing fungicide use and combating resistance against B. cinerea.

Colonization of legumes by an endophytic Fusarium solani strain FsK reveals common features to symbionts or pathogens.

Plant cellular responses to endophytic filamentous fungi are scarcely reported, with the majority of described colonization processes in plant-fungal interactions referring to either pathogens or true symbionts. Fusarium solani strain K (FsK) is a root endophyte of Solanum lycopersicum, which protects against root and foliar pathogens. Here, we investigate the association of FsK with two legumes (Lotus japonicus and Medicago truncatula) and report on colonization patterns and plant responses during the establishment of the interaction. L. japonicus plants colonized by FsK complete their life cycle and exhibit no apparent growth defects under normal conditions. We followed the growth of FsK within root-inoculated plants spatiotemporally and showed the capability of the endophyte to migrate to the stem. In a bipartite system comprising of the endophyte and either whole plants or root organ cultures, we studied the plant sub-cellular responses to FsK recognition, using optical, confocal and transmission electron microscopy. A polarized reorganization of the root cell occurs: endoplasmic reticulum/cytoplasm accumulation and nuclear placement at contact sites, occasional development of papillae underneath hyphopodia and membranous material rearrangements towards penetrating hyphae. Fungal hyphae proliferate within the vascular bundle of the plant. Plant cell death is involved in fungal colonization of the root. Our data suggest that the establishment of FsK within legume tissues requires fungal growth adaptations and plant cell-autonomous responses, known to occur during both symbiotic and pathogenic plant-fungal interactions. We highlight the overlooked plasticity of endophytic fungi upon plant colonization, and introduce a novel plant-endophyte association.

Use of copper, silver and zinc nanoparticles against foliar and soil-borne plant pathogens.

Nano-fungicides are expected to play an important role in future plant disease management as eco-friendly alternatives of conventional synthetic fungicides. In the present study, the sensitivity of seven fungal species, known to cause foliar and soil-borne diseases, to nanoparticles (NPs) containing copper (Cu-NPs, CuO-NPs), silver (Ag-NPs) and zinc (ZnO-NPs) was assessed in vitro. Mycelial growth assays revealed that Cu-NPs with mean inhibition rates, EC50, ranging between 162 and 310 µg/mL were most effective among the NPs tested in inhibiting fungal growth, followed by ZnO-NPs with EC50 ranging between 235 and 848 µg/mL. All fungal species were practically insensitive to CuO-NPs and Ag-NPs except for B. cinerea, which was equally sensitive to Ag-NPs and Cu-NPs (EC50 = 307 µg/mL). Cu-NPs were more fungitoxic in terms of mycelial growth, to almost all species tested, than a protective fungicide containing Cu(OH)2, which was used as a reference. Fungitoxicity experiments with the NPs tested and bulk size reagents containing the respective metals revealed that ZnO-NPs were more toxic to all fungal species tested than ZnSO4, whereas Cu-NPs were more fungitoxic than CuSO4 in all cases, except for B. cinerea, A. alternata and M. fructicola. The existence of a positive correlation between Cu-NPs and CuO-NPs toxicity and, at the same time, the absence of any correlation between NPs tested and their respective bulk metal counterparts indicated potential differences in the mode of action between bulk and nanosized antifungal ingredients. Although there was considerable variation between fungal species, all NPs were generally 10 to 100 fold more fungitoxic to spores than hyphae and in the majority of cases more effective than Cu(OH)2, as revealed by colony formation bioassays. NPs significantly suppressed grey mold symptoms on plum fruit, especially Ag-NPs, which completely inhibited disease development. Consequently, tested NPs have the potential to be used as protective antifungal agents.

The Beneficial Endophytic Fungus Fusarium solani Strain K Alters Tomato Responses Against Spider Mites to the Benefit of the Plant.

Beneficial microorganisms are known to promote plant growth and confer resistance to biotic and abiotic stressors. Soil-borne beneficial microbes in particular have shown potential in protecting plants against pathogens and herbivores via the elicitation of plant responses. In this study, we evaluated the role of Fusarium solani strain K (FsK) in altering plant responses to the two spotted spider mite Tetranychus urticae in tomato. We found evidence that FsK, a beneficial endophytic fungal strain isolated from the roots of tomato plants grown on suppressive compost, affects both direct and indirect tomato defenses against spider mites. Defense-related genes were differentially expressed on FsK-colonized plants after spider mite infestation compared to clean or spider mite-infested un-colonized plants. In accordance, spider mite performance was negatively affected on FsK-colonized plants and feeding damage was lower on these compared to control plants. Notably, FsK-colonization led to increased plant biomass to both spider mite-infested and un-infested plants. FsK was shown to enhance indirect tomato defense as FsK-colonized plants attracted more predators than un-colonized plants. In accordance, headspace volatile analysis revealed significant differences between the volatiles emitted by FsK-colonized plants in response to attack by spider mites. Our results highlight the role of endophytic fungi in shaping plant-mite interactions and may offer the opportunity for the development of a novel tool for spider mite control.

A Fusarium solani endophyte vs fungicides: Compatibility in a Fusarium oxysporum f.sp. radicis-lycopersici - tomato pathosystem.

The potential of FsK, a non-pathogenic endophytic Fusarium solani strain, to be utilized as a biocontrol agent in combination with nine selected fungicides registered in tomato crops in Greece was evaluated. In vitro fungitoxicity tests revealed that FsK was insensitive to doses exceeding 100 µg/mL of thiophanate-methyl, fenhexamid, cyprodinil, boscalid and mancozeb. On the contrary, prochloraz, fludioxonil, pyraclostrobin and difenoconazole were most toxic to FsK. None of the later fungicides affected conidial production in an adverse way. Drenching of tomato plants with the above fungicides at recommended doses did not significantly affect colonization of tomato roots by FsK as revealed by in vitro isolation and Real Time PCR quantification. The disease suppressive ability of FsK against Fusarium oxysporum f.sp.radicis lycopersici (FORL) was not adversely affected by the post-inoculation application of commercial formulations of fludioxonil (Switch) and pyraclostrobin (Comet) at the recommended doses. Even more, the Comet-FsK combination resulted in enhanced disease suppression compared to either of the two treatments applied individually. In conclusion, not only biocontrol agent FsK is suitable for use in tomato integrated disease management programs that include all tested fungicides but also, some FsK -fungicide combinations can have additive effect against FORL disease incidence.

Characterization of Fungi Associated With Wood Decay of Tree Species and Grapevine in Greece.

A two-year survey was conducted to identify fungi associated with wood decay in a range of tree species and grapevine. Fifty-eight fungal strains isolated from plants of 18 species showing typical wood decay symptoms were characterized by morphological, physiological, and molecular analyses. By 5.8S rRNA gene-ITS sequencing analysis, these isolates were classified into 25 distinct operational taxonomic units, including important phytopathogenic species of the phyla Pezizomycotina and Agaricomycotina, such as Fomitiporia, Inonotus, Phellinus, Inocutis, Fuscoporia, Trametes, Fusarium, Eutypa, Phaeomoniella, Phaeoacremonium, and Pleurostomophora spp. The white rot basidiomycetes Fomitiporia mediterranea (20 isolates, 34.5%) and Inonotus hispidus (6 isolates, 10.3%) were the most prevalent. Pathogenicity tests revealed for the first time that certain fungal species of the genera Fomitiporia, Inonotus, Phellinus, Pleurostomophora, and Fusarium caused wood infection of various tree species in Greece and worldwide. To the best of our knowledge, this is the first report of F. mediterranea as the causal agent of wood decay in pear, pomegranate, kumquat, and silk tree. This is also the first record of Inonotus hispidus, Phellinus pomaceus, Pleurostomophora richardsiae, and Fusarium solani in apple, almond, avocado, and mulberry tree, respectively, whereas P. richardsiae was associated with wood infection of olive tree for the first time in Greece. Cross pathogenicity tests with F. mediterranea strains originated from grapevine applied on other woody hosts and from olive on grapevine demonstrated partial host specificity of the fungus. The potential of F. mediterranea to transinfect hosts other than those originated, along with the host range extension of the fungus, is discussed.

Role of lupeol synthase in Lotus japonicus nodule formation.

• Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. • The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. • The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. • The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression.

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.

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.