Survey of root rot diseases of sugar bett in Central Greece

An extensive survey was conducted during the summer and autumn of 2004 in sugar beet fields in the area of Larissa, Thessaly region, with plants showing symptoms of root rot diseases. The aim of the monitoring was to identify the causal agents of root rot diseases. In total, 76 sugar beet fields were surveyed and 5-10 diseased roots were examined from each field. Isolations, carried out on PDA, showed that two main fungal pathogens causing root rot were Rhizoctonia solani and Phytophthora cryptogea. The former was isolated in 46% of the fields and the latter in 38% of the fields. In addition, Rhizopus stolonifer, Fusarium spp., Scerotium rolfsii and Rhizoctonia violacea were isolated in 14%, 7%, 4% and 1% of the fields respectively. In most of the surveyed fields only one pathogen species was isolated and only in a few of them more than one fungal species was identified

Insights into the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, the pathogen Botrytis cinerea and their plant host

Botrytis cinerea is a plant pathogen causing the gray mold disease in a plethora of host plants. The control of the disease is based mostly on chemical pesticides, which are responsible for environmental pollution, while they also pose risks for human health. Furthermore, B. cinerea resistant isolates have been identified against many fungicide groups, making the control of this disease challenging. The application of biocontrol agents can be a possible solution, but requires deep understanding of the molecular mechanisms in order to be effective. In this study, we investigated the multitrophic interactions between the biocontrol agent Bacillus subtilis MBI 600, a new commercialized biopesticide, the pathogen B. cinerea and their plant host. Our analysis showed that this biocontrol agent reduced B. cinerea mycelial growth in vitro, and was able to suppress the disease incidence on cucumber plants. Moreover, treatment with B. subtilis led to induction of genes involved in plant immunity. RNAseq analysis of B. cinerea transcriptome upon exposure to bacterial secretome, showed that genes coding for MFS and ABC transporters were highly induced. Deletion of the Bcmfs1 MFS transporter gene, using a CRISP/Cas9 editing method, affected its virulence and the tolerance of B. cinerea to bacterial secondary metabolites. These findings suggest that specific detoxification transporters are involved in these interactions, with crucial role in different aspects of B. cinerea physiology

Fusarium equiseti as an Emerging Foliar Pathogen of Lettuce in Greece: Identification and Development of a Real-Time PCR for Quantification of Inoculum in Soil Samples

Lettuce is the most commonly cultivated leafy vegetable in Greece, available in the market throughout the year. In this study, an emerging foliar disease observed in commercial farms has been associated to the pathogen Fusarium equiseti, a member of the Fusarium incarnatum-equiseti species complex (FIESC). Thirty F. equiseti isolates obtained from symptomatic lettuce plants were identified on the basis of morphology and evaluated for their pathogenicity. The isolates were further characterized using amplification and sequence analysis of the internal transcribed region (ITS-rDNA), and of the translation elongation factor 1-alpha (TEF1-a), calmodulin (CAM), beta-tubulin (Bt), and small subunit (SSU) genes. Moreover, a novel RT-qPCR assay was developed, designing a primer pair and a probe based on the TEF1-a sequences. This assay showed high specificity, amplifying F. equiseti DNA samples, while no amplification product was observed from samples of other common soilborne fungi. The generated RT-qPCR assay could be a useful tool for the detection and quantification of F. equiseti in soil samples deriving from fields cultivated with lettuce and other leafy vegetables, hosts of this specific pathogen

Laminarin Induces Defense Responses and Efficiently Controls Olive Leaf Spot Disease in Olive

Olive leaf spot (OLS) caused by Fusicladiumoleagineum is mainly controlled using copper fungicides. However, the replacement of copper-based products with eco-friendly alternatives is a priority. The use of plant resistance-inducers (PRIs) or biological control agents (BCAs) could contribute in this direction. In this study we investigated the potential use of three PRIs (laminarin, acibenzolar-S-methyl, harpin) and a BCA (Bacillus amyloliquefaciens FZB24) for the management of OLS. The tested products provided control efficacy higher than 68%. In most cases, dual applications provided higher (p < 0.05) control efficacies compared to that achieved by single applications. The highest control efficacy of 100% was achieved by laminarin. Expression analysis of the selected genes by RT-qPCR revealed different kinetics of induction. In laminarin-treated plants, for most of the tested genes a higher induction rate (p < 0.05) was observed at 3 days post application. Pal, Lox, Cuao and Mpol were the genes with the higher inductions in laminarin-treated and artificially inoculated plants. The results of this study are expected to contribute towards a better understanding of PRIs in olive culture and the optimization of OLS control, while they provide evidence for potential contributions in the reduction of copper accumulation in the environment

Effect of ozone application during cold storage of kiwifruit on the development of stem-end rot caused by Botrytis cinerea

The effect of gaseous ozone exposure on the development of stem-end rot disease, caused by Botrytis cinerea, on kiwifruit (Actinidia deliciosa, cv. Hayward) was investigated. Artificially inoculated kiwifruit were subjected for 4 months to conventional cold storage (0 °C, RH 95%) where catalytic oxidation of ethylene was applied (control) and to conventional cold storage with continuous supply of ozone (0.3 μL L−1) or in a conventional kiwifruit cold storage room, where catalytic oxidation of ethylene was applied. Ozone treatment delayed and simultaneously decreased disease incidence by 56%, while disease severity on infected fruit remained unaffected. Infected fruit formed sclerotia, while no sporulation of the pathogen occurred in the presence of ozone. To elucidate whether the observed disease suppression was mediated by a direct effect of ozone on the fungal pathogen per se or by the induction of a resistance mechanism in the fruit, two additional sets of experiments were conducted. Kiwifruit were exposed to ozone (0.3 μL L−1) for 0, 2, 8, 24, 72 and 144 h in a conventional cold storage room either before or after the artificial inoculation with the pathogen and its efficacy on disease incidence and severity was monitored. Pre-inoculation exposure of fruit to ozone, at increasing exposure time intervals led to significant suppression of disease incidence, while post-inoculation exposure did not affect it. The observed disease suppression, provided by the pre-inoculation exposure, strongly suggests that ozone treatments induce resistance of kiwifruit to the pathogen. Measurements of antioxidant substances and antioxidant activity on fruit exposed to ozone for the same time intervals showed a strong negative correlation between disease incidence or severity and phenol content

Identification and mycotoxigenic capacity of fungi associated with pre- and postharvest fruit rots of pomegranates in Greece and Cyprus

Pre- and postharvest fruit rots of fungal origin are an important burden for the pomegranate industry worldwide, affecting the produce both quantitatively and qualitatively. During 2013, local orchards were surveyed and 280 fungal isolates from Greece (GR) and Cyprus (CY) were collected from pomegranates exhibiting preharvest rot symptoms, and additional 153 isolates were collected postharvest from cold-stored fruit in GR. Molecular identification revealed that preharvest pomegranate fruit rots were caused predominately by species of the genera Aspergillus (Aspergillus niger and Aspergillus tubingensis) and Alternaria (Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens). By contrast, postharvest fruit rots were caused mainly by Botrytis spp. and to a lesser extent by isolates of Pilidiella granati and Alternaria spp. Considering that a significant quota of the fungal species found in association with pomegranate fruit rots are known for their mycotoxigenic capacity in other crop systems, their mycotoxin potential was examined. Alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) production was estimated among Alternaria isolates, whereas ochratoxin A (OTA) and fumonisin B2 (FB2) production was assessed within the black aspergilli identified. Overall in both countries, 89% of the Alternaria isolates produced AOH and AME in vitro, while TEN was produced only by 43.9%. In vivo production of AOH and AME was restricted to 54.2% and 31.6% of the GR and CY isolates, respectively, while none of the isolates produced TEN in vivo. Among black aspergilli 21.7% of the GR and 17.8% of the CY isolates produced OTA in vitro, while in vivo OTA was detected in 8.8% of the isolates from both countries. FB2 was present in vitro in 42.0% of the GR and 22.2% of the CY isolates, while in vivo the production was limited to 27.5% and 4.5% of the GR and the CY isolates, respectively. Our data imply that mycotoxigenic Alternaria and Aspergillus species not only constitute a significant subset of the fungal population associated with pomegranate fruit rots responsible for fruit deterioration, but also pose a potential health risk factor for consumers of pomegranate-based products

Synthesis and Characterization of Novel Copper Nanoparticles for the Control of Leaf Spot and Anthracnose Diseases of Olive

Olive crop is frequently treated with copper fungicides to combat foliar and fruit diseases such as olive leaf spot caused by Fusicladium oleagineum and anthracnose caused by Colletotrichum spp. The replacement of copper-based products with more eco-friendly alternatives is a priority. Metal nanoparticles synthesized in several ways have recently revolutionized crop protection with applications against important crop pathogens. In this study, we present the development of four copper-based nanoparticles (CuNP Type 1 to 4) synthesized with a wet chemistry approach. The CuNPs were characterized using Transmission Electron Microscopy, Dynamic Light Scattering, Laser Doppler Electrophoresis, and Attenuated Total Reflection measurements. In addition, the activity of the four CuNP types was tested in vitro and in planta against F. oleagineum and Colletotrichum spp. In vitro sensitivity measurements showed that for both pathogens, mycelial growth was the most susceptible developmental stage to the tested compounds. Against both pathogens, CuNP Type 1 and Type 2 were found to be more active in reducing mycelial growth compared to the reference commercial compounds of copper oxide and copper hydroxide. In planta experiments showed that CuNP Type 3 and CuNP Type 4 exhibited a strong protectant activity against both F. oleagineum and Colletotrichum acutatum with control efficacy values significantly higher than those achieved by the applications of either reference product

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

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

Development of a Microwave-Assisted Extraction Protocol for the Simultaneous Determination of Mycotoxins and Pesticide Residues in Apples by LC-MS/MS

The possible presence of contaminants, pesticide residues and mycotoxins, in agricultural commodities is a critical issue for food safety, causing great concern. In this work, a simple and rapid analytical method employing liquid chromatography–tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of pesticide residues and mycotoxins in apples. Microwave-assisted extraction (MAE) was used for sample preparation. The MAE protocol was optimized after evaluating the effects of the following extraction parameters: (a) extraction solvent, (b) acidic environment, (c) temperature, and (d) extraction time. The multiresidue MAE-LC-MS/MS method was validated for linearity, accuracy (bias and precision), limits of detection (LODs), limits of quantification (LOQs), and matrix effect. The validation of the method was carried out according to the SANTE/12682/2019 document. The method demonstrated good linearity with R2 ≥ 0.99, acceptable accuracy in the recovery rate range 70–116%, acceptable interassay precision with RSD% ≤ 20, and low LODs and LOQs in the ranges 0.005–0.015 μg/g and 0.01–0.03 μg/g, respectively. Matrix effects were observed only for the 25% of the analytes. The performance of the MAE-LC-MS/MS method was compared to that of the QuEChERS sample preparation method, and the MAE-LC-MS/MS method proved to be rapid and effective