This study investigated the impact of metallic nanoparticles (NPs) containing copper, silver, copper oxide, and zinc oxide, recognized as potential pollutants, on the structural and compositional aspects of soil microbial communities in comparison to their bulk counterparts. The influence of these nanoparticles was examined at two distinct accumulation levels within the soil ecosystem. The potential effects of metallic nanoparticles in comparison to their bulk counterparts were evaluated in a pot experiment under controlled environmental conditions. High-throughput sequencing of PCR-amplified 16S rRNA and ITS2 marker genes was employed to analyze the impact of NPs and counterparts on bacterial and fungal rhizospheric communities using two dosage levels. Bioinformatic analysis of the obtained sequencing results revealed a distinct metal-dependent differentiation in bacterial and fungal soil community structures. Silver-containing treatments exhibited an enhanced ability to induce changes in both bacterial and fungal communities compared to other metals. Furthermore, treatment dose had a profound differentiation effect on the two microbial communities. The low dose notably influenced bacterial communities to a greater extent compared to the high dose, whereas fungal communities exhibited significant alterations under high-dose conditions rather than under low-dose conditions. This study was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Rural Development Program (RDP/ΠΑΑ) 2014 – 2020, under the call "Cooperation for environmental projects, environmental practices and actions for climate change” (project code: Μ16SΥΝ2-00354) and by the European Union- Next Generation EU, Greece 2.0 National Recovery and Resilience plan (project code: TAEDR-0535675)

The effectiveness of copper containing nanoparticles (Cu/CuO-NPs) against insecticide-resistant olive fruit flies (Bactrocera oleae) and their impact on the insect’s reproductive and endosymbiotic parameters were evaluated. The insecticidal activity of both nano and bulk copper [Cu(OH)2] was comparable or greater than that of the reference insecticide deltamethrin at recommended doses as revealed by feeding experiments. A significant synergistic effect between Cu-NPs or CuO-NPs and deltamethrin was observed in terms of adult mortality. Furthermore, the deltamethrin + Cu-NPs combination decreased the total number of offspring as compared with the untreated control. The above combination also significantly decreased the mean number of stings, pupae, female and total number offspring of the surviving female, compared to deltamethrin applied alone. The abundance of the Candidatus Erwinia dacicola- a B. oleae larvae bacterial gut endosymbiont- was adversely affected by bulk and nanosized copper. Concluding, Cu-NPs have a great potential to control insecticide-resistant B. oleae populations by reducing adult and larval survival and fecundity, and provide the means for reducing the environmental footprint of pesticides by minimizing their required doses. This study was co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Rural Development Program (RDP/ΠΑΑ) 2014 – 2020, under the call "Cooperation for environmental projects, environmental practices and actions for climate change“ (project code: Μ16SΥΝ2-00354).

In September 2023, thirty declining 30-year-old avocado (Persea americana) trees ('Hass' grafted on 'Zutano' seedlings) were detected in a 1.5-ha orchard in the island of Crete (Chania region). Crown symptoms encompassed wilting and leaf chlorosis, advancing to defoliation and extensive dieback. Tap and feeder roots decayed and brown discoloration of root tissues was evident on heavily infected trees. The disease was severe and widespread, resulting in a 5% mortality rate among 300 trees. The pathogen was isolated with a modified soil baiting technique (Ferguson and Jeffers, 1999). Surface disinfected avocado fruits were immersed in water containing soil samples. Following a period of 2 to 8 days, tissue fragments from the resulting necrotic lesions on the fruit surface were transferred on ΡΑRP medium and subsequently incubated at 20°C (Ferguson and Jeffers, 1999). Three isolates (AV2, AV12 and AV11a) were obtained by transferring single hyphal tips to new Petri dishes containing V8 juice agar. They were grown at 20˚C and used for identification after 10 days. Isolates formed coralloid colonies with abundant clustered spherical hyphal swellings and terminal or intercalary (ratio 1:5) thick-walled chlamydospores measuring 20 to 36 μm (avg 29±0.8 μm) with characteristic thick walls (avg 1.2±0.2 μm). Sporangia, produced in non-sterile soil-extract water, were ovoid to obpyriform, persistent, non-papillate, 32 to 81 μm (avg 56±4.8 μm) long and 20 to 42 μm (avg 31±3.2 μm) wide (n=100). Isolates were heterothallic as they did not produce oospores in single cultures. Based on the morphological traits the isolates were identified as Phytophthora cinnamomi (Erwin and Ribeiro 1996). The internal transcribe spacer region (ITS) including ITS1, 5.8S rDNA region, and ITS2 as well as the cytochrome c oxidase subunit I (coxI) gene of the three representative isolates wereamplified with ITS1/ITS4 and FM83/FM84 primers, respectively (White et al. 1990; Martin and Tooley 2003), and sequenced (GenBank acc. PP506613 to PP506615 and PQ063867 to PQ063869, respectively). BLAST search revealed almost 100% identity with the sequences of P. cinnamomi ex-isotype isolate (KC478663 and KU899315 respectively). Pathogenicity tests using isolate AV2 were conducted following the soil infestation method (Jung et al. 1996) using six-year-old avocado 'Zutano' seedlings. Six non-inoculated plants treated with vermiculite-multivitamin juice mixture were used as controls. Plants (1 m tall) were grown in pots under greenhouse conditions and watered regularly. Six weeks post inoculation, all inoculated trees showed chlorosis, wilting and root rot, while control plants remained symptomless. Symptoms were similar to those observed in the field and the pathogen was re-isolated and molecularly identified as previously described. This study presents the first documented occurrence of P. cinnamomi, widely regarded as the most destructive avocado pathogen globally, on avocado crops in Greece (Rodger et al. 2019). Additionally, this marks the first recorded presence of this pathogen on the island of Crete, regardless of the host species. The accurate identification of Phytophthora species associated with avocado root rot is essential for implementing an effective disease management strategy, particularly in the selection of appropriate disease-resistant rootstocks.

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.

The potential of ZnO nanoparticles (NPs) to control Alternaria alternata isolates resistant to the succinate dehydrogenase inhibitor (SDHI) boscalid was evaluated both in vitro and in vivo. ZnONPs could effectively inhibit mycelial growth and suppress disease symptoms in both boscalid sensitive (BOSC-S) and resistant (BOSC-R) isolates. A high synergistic effect against BOSC-S and BOSC-R isolates was observed when ZnO-NPs was combined with boscalid both in vitro and when applied in artificially inoculated tomato fruit. The positive correlation between nanoparticles and their ionic counterpart ZnSO4 and the neutralization of the ZnO-NPs fungitoxic action in the presence of EDTA suggested that zinc ion release is the most probable fungitoxic mechanism of ZnO-NPs. The disruption of cellular ion homeostasis mechanisms by zinc NPs could account for the enhanced effectiveness of ZnO-NPs against A. alternata compared to ZnSO4. ATP-dependent ion efflux and ROS production could contribute to the fungitoxic action of ZnO-NPs as indicated by bioassays with ATP- and antioxidant-inhibitors. Boscalid acting as a “capping” agent for ZnO-NPs, significantly reducing NPs mean size, probably accounted for the synergy observed against BOSC-S and BOSC-R isolates. Concluding, ZnO-NPs are effective against A. alternata both alone or in mixtures with boscalid, and can be used as an effective, eco-compatible anti-resistance tool for reducing the environmental footprint of synthetic fungicides.

A recent increase in the Calocoris trivialis populations associated with premature olive blossom dropping in Crete coupled with ineffective control by organophosphate and pyrethroid insecticides resulted in resistance development speculation. A stem-dip assay was developed to evaluate C. trivialis susceptibility to dimethoate and beta-cyfluthrin against nymphal and adult insect stages. Susceptibility of C. trivialis populations collected from two Cretan orchards with no (AGRK) and extensive spray history (SPL) to both insecticides was evaluated and compared. Dimethoate LC50 values of the AGRK population were 17.47 and 33.37 mg L−1 for adults and nymphs and 45.85 and 20.08 mg L−1 for SPL respectively. C. trivialis was significantly more susceptible to beta-cyfluthrin as indicated by LC50 values for AGRK, SPL adults (0.53, 0.73 mg L−1) and nypmphs (1.44, 2.21 mg L−1). No evidence for resistance development was found in any of the insecticide cases. Adult insects were significantly more sensitive than nymphs in both insecticide/population combinations. Ratios of nymph: adult LC50values were 1.91, 2.71 for dimethoate and beta-cyfluthrin in AGRK and 2.28, 3.02 in SPL population. Susceptibility data and evidence of differential stage-dependent response of C. trivialis to both insecticides should be taken account for the establishment of effective control strategies.

The potential of silver nanoparticles (Ag-NPs) to control plant pathogen Moniliafructicola and to deter environmental contamination by reducing fungicide doses was evaluated in vitro and in vivo. Fungitoxicity screening of M. fructicola isolates resulted in the detection of 18 benzimidazole-resistant (BEN-R) isolates with reduced sensitivity to fungicides thiophanate methyl (TM) and carbendazim. All resistant isolates caried the E198A resistance mutation in their β-tubulin gene, target site of the benzimidazole fungicides. Ag-NPs could effectively control both sensitive (BEN-S) and resistant isolates while the combination of Ag-NPs with TM significantly enhanced their fungitoxic effect both in vitro and in apple fruit tests. The positive correlation observed between Ag-NPs and TM+Ag-NPs treatments indicates a mixture-enhanced Ag-NPs activity/availability as a possible mechanism of synergy. No correlation between Ag-NPs and AgNO3 could be found suggesting difference(s) in the fungitoxic mechanism of action between Nps and their bulk/ionic counterparts. Indications of the involvement of energy (ATP) metabolism in the mode of action of Ag-NPs were also evident by the synergy observed between Ag-NPs and the oxidative phosphorylation-uncoupler fluazinam (FM) against both BEN-R and BEN-S phenotypes. The role of silver ions release on the inhibitory action of Ag-NPs against the fungusis probably limited since the AgNPs/NaCl combination enhanced fungitoxicity, a fact that could not be justified by the expected binding of silver with chlorine ions. Concluding, Ag-NPs can be effectively used as a means of controlling both BEN-S and BEN-R M. fructicola isolates while their combination with conventional fungicides should aid anti-resistant strategies and reduce the environmental impact of synthetic fungicides by reducing effective doses to the control the pathogen.

The potential of copper nanoparticles (Cu-NPs) alone or in combination with conventional fungicides against sensitive and resistant to fungicides Botrytis cinerea isolates was assessed in vitro and in vivo. DNA sequencing revealed the E198A resistance mutation in the β-tubulin gene in three B. cinerea isolates highly resistant to benzimidazoles (BEN-R), thiophanare methyl (TM), and the G143A mutation in the cytb gene in four isolates highly resistant to the QoI pyraclostrobin (PYR-R). Cu-NPs could effectively control sensitive and resistant isolates. A synergistic effect between Cu-NPs and TM both in vitro and in vivo was observed in the case of benzimidazole sensitive isolates while an additive effect was observed in BEN-R isolates. The above observed synergistic action could be attributed to increased TM availability in the target site as indicated by the positive correlation observed between TM and TM+Cu-NPs treatments. A positive cross sensitivity and antagonistic action between Cu-NPs and NaCl suggested that copper ions contribute in the fungitoxic action of Cu-NPs, at least partly, since no correlation between Cu(OH)2 and Cu-NPs sensitivity was found. The co-application of Cu-NPs with the oxidative phosphorylation inhibitor fluazinam (FM) resulted in a synergistic action in all isolates regardless resistance phenotype, indicating a ATP-dependent mechanism of toxic action of Cu-NPs. Cu-NPs combined with conventional fungicides can aid in the design and implementation of eco friendly, sustainable management strategies by reducing fungicide use and combating resistance against B. cinerea.

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 mu g/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 Esp.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. (C) 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Fenhexamid is a hydroxyanilide fungicide with excellent performance against Botrytis cinerea but also effective against Monilinia spp. which cause brown rot disease in apple and stone fruit. A total of 75 Monilinia laxa field isolates were utilized to determine baseline sensitivity while a number of fenhexamid-resistant laboratory mutants were used to evaluate the resistance risk associated with the longevity of the effectiveness of fenhexamid. Fenhexamid was found to be highly effective against all field isolates. EC50 values ranged from 0.02 to 1 mu g mL(-1) and were distributed unimodaly around an average of 0.1 mu g mL(-1). M. laxa laboratory strains with moderate and high resistance levels to fenhexamid were isolated after UV mutagenesis. All fenhexamid-resistant strains showed parental sensitivity to carbendazim, iprodione, fludioxonil, pyraclostrobin, flusilazole and prochloraz. Interestingly, some of the mutant strains were also resistant to tridemorph, fenpropimorph and spiroxamine. Studies on fitness parameters of fenhexamid-resistant strains revealed a fitness cost on sporulation and pathogenicity but not on mycelial growth. These results suggest that fenhexamid should be a good alternative site-specific fungicide for the control of brown rot disease caused by M. laxa. However, appropriate anti-resistance strategies should be considered to ensure the successful commercial use of fenhexamid in the long run. (c) 2012 Elsevier Ltd. All rights reserved.