Identification of Fusarium oxysporum f. sp. lactucae Race 1 as the Causal Agent of Lettuce Fusarium Wilt in Greece, Commercial Cultivars' Susceptibility, and Temporal Expression of Defense-Related Genes.

Fusarium wilt of lettuce is found throughout the world, causing significant yield losses. Lettuce is the most-cultivated leafy vegetable in Greece, affected by a large number of foliar and soil-borne pathogens. In this study, 84 isolates of Fusarium oxysporum, obtained from soil-grown lettuce plants exhibiting wilt symptoms, were characterized as belonging to race 1 of F. oxysporum f. sp. lactucae based on sequence analysis of the translation elongation factor 1-alpha (TEF1-α) gene and the rDNA intergenic spacer (rDNA-IGS) region. The isolates were also assigned to one single race through PCR assays with specific primers targeting race 1 and race 4 of the pathogen. In addition, four representative isolates were confirmed to be associated with race 1 based on the pathogenicity tests with a set of differential lettuce cultivars. Artificial inoculations on the most commonly cultivated lettuce cultivars in Greece revealed that the tested cultivars varied regarding their susceptibility to F. oxysporum f. sp. lactucae race 1. Cultivars (cvs.) "Cencibel" and "Lugano" were found to be highly susceptible, while cvs. "Sandalina" and "Starfighter" were the most resistant ones. Expression analysis of 10 defense-related genes (PRB1, HPL1, LTC1, SOD, ERF1, PAL1, LOX, MPK, BG, and GST) was carried out on artificially inoculated lettuce plants of the four above cultivars at different time points after inoculation. In resistant cultivars, a higher induction rate was observed for all the tested genes in comparison with the susceptible ones. Moreover, in resistant cultivars, all genes except LTC1, MPK, and GST showed their highest induction levels in their earliest stages of infection. The results of this study are expected to contribute to the implementation of an integrated management program to control Fusarium wilt of lettuce, based mainly on the use of resistant cultivars.

Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

Resistance of Black Aspergilli Species from Grape Vineyards to SDHI, QoI, DMI, and Phenylpyrrole Fungicides.

Fungicide applications constitute a management practice that reduces the size of fungal populations and by acting as a genetic drift factor, may affect pathogen evolution. In a previous study, we showed that the farming system influenced the population structure of the Aspergillus section Nigri species in Greek vineyards. The current study aimed to test the hypothesis that the differences in the population structure may be associated with the selection of fungicide-resistant strains within the black aspergilli populations. To achieve this, we determined the sensitivity of 102, 151, 19, and 22 for the A. uvarum, A. tubingensis, A. niger, and A. carbonarious isolates, respectively, originating either from conventionally-treated or organic vineyards to the fungicides fluxapyroxad-SDHIs, pyraclostrobin-QoIs, tebuconazole-DMIs, and fludioxonil-phenylpyrroles. The results showed widespread resistance to all four fungicides tested in the A. uvarum isolates originating mostly from conventional vineyards. In contrast, all the A. tubingensis isolates tested were sensitive to pyraclostrobin, while moderate frequencies of only lowly resistant isolates were identified for tebuconazole, fludioxonil, and fluxapyroxad. Sequencing analysis of the corresponding fungicide target encoding genes revealed the presence of H270Y, H65Q/S66P, and G143A mutations in the sdhB, sdhD, and cytb genes of A. uvarum resistant isolates, respectively. No mutations in the Cyp51A and Cyp51B genes were detected in either the A. uvarum or A. tubingensis isolates exhibiting high or low resistance levels to DMIs, suggesting that other resistance mechanisms are responsible for the observed phenotype. Our results support the initial hypothesis for the contribution of fungicide resistance in the black aspergilli population structure in conventional and organic vineyards, while this is the first report of A. uvarum resistance to SDHIs and the first documentation of H270Y or H65Q/S66P mutations in sdhB, sdhD, and of the G143A mutation in the cytb gene of this fungal species.

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.

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.

Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L-1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L-1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

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.

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. RNA-seq 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.

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.

Characterization of boscalid-resistance conferring mutations in the SdhB subunit of respiratory complex II and impact on fitness and mycotoxin production in Penicillium expansum laboratory strains.

Laboratory mutants of Penicillium expansum highly resistant (Rfs: 90 to >500, based on EC50s) to Succinate Dehydrogenase Inhibitors (SDHIs) were isolated after UV-mutagenesis and selection on media containing boscalid. A positive correlation was found between sensitivity of isolates to boscalid and other SDHIs such as isopyrazam and carboxin but not to fungicides affecting other cellular pathways or processes, such as the triazole flusilazole, the phenylpyrrole fludioxonil, the anilinopyrimidine cyprodinil and the benzimidazole benomyl. Most of the boscalid-resistant strains were more sensitive to the SDHI fluopyram and the QoI pyraclostrobin. In order to investigate the mechanism responsible for the observed resistance profiles, part of the SdhB subunit isolated the wild type and boscalid-resistant isolates, was genetically characterized. Comparison of the deduced amino-acid sequence between resistant and wild-type isolates revealed two point mutations at a position corresponding to codon 272 of the respective SdhB protein in Botrytis cinerea. The substitution of histidine by arginine was found in boscalid-resistant isolates which were equally sensitive to fluopyram compared with the wild-type whereas the replacement of histidine by tyrosine was found in strains with increased sensitivity to fluopyram. No adverse effects of resistance mutations were observed on fitness determining parameters such as osmotic sensitivity, sporulation and pathogenicity, while mycelial growth rate and spore germination was negatively affected in some of the mutants studied. P. expansum mutant strains displayed significantly perturbed patulin and citrinin levels as compared to the wild-type parent strain both in vitro and in vivo as revealed by thin layer (TLC) and high performance liquid chromatography (HPLC).

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis.

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using High-Resolution Melting (HRM) Analysis.

Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit.

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.

Determination of mycotoxins in pomegranate fruits and juices using a QuEChERS-based method.

A rapid and accurate analytical method for the determination of three Alternaria mycotoxins (alternariol, alternariol monomethyl ether, and tentoxin) in pomegranate samples (fruits and juices) was developed and validated. The overall average recoveries ranged for 82.0-109.4% and the relative standard deviations were from 1.2% to 10.9%. The optimized and validated method was applied to detect the presence of the target mycotoxins in real samples (fruits and juices) purchased from Greek markets. Mycotoxins were not found in any of the analyzed samples. Also, artificially inoculated pomegranate fruits with six different Alternaria alternata species complex isolates, known to produce the target mycotoxins on pure cultures, were analyzed and alternariol concentrations found ranged from 0.3 to 50.5 µg/g, alternariol monomethyl ether from 0.5 to 32.3 µg/g, while tentoxin was not detected. The developed analytical method can be used for the routine monitoring of the major Alternaria mycotoxins in pomegranates.

Identification, characterization and mycotoxigenic ability of Alternaria spp. causing core rot of apple fruit in Greece.

Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece. The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressiveness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequencies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial inoculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation at two different temperatures (2 and 25°C) showed that fruit of Fuji variety were the most susceptible and fruit of Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species. Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially inoculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD). The results showed that most of the isolates of both species were able to produce all the 3 metabolites both in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of apple by-products.

Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum.

Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the ß-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the ß-tubulin gene in moderately resistant isolates did not reveal any amino acid substitution. This is the first report on the existence and distribution of highly mycotoxigenic field isolates of P. expansum resistant to the benzimidazoles indicating a high potential risk of increased mycotoxin contamination of pome fruit and by-products.

Differential Effect of SdhB Gene Mutations on the Sensitivity to SDHI Fungicides in Botrytis cinerea.

Succinate dehydrogenase inhibiting (SDHI) fungicides constitute a relatively novel fungicide group used for gray mold control caused mainly by Botrytis cinerea. Shortly after registration, resistance was observed in fungal populations that correlated with several mutations in the succinate dehydrogenase complex (complex II). In the current study, 30 B. cinerea isolates possessing five different mutations at three different codons of SdhB (P225F, N230I, and H272L/R/Y) were characterized for their sensitivities to eight SDHI fungicides. The results show different sensitivities and cross-resistance patterns between structurally different SDHIs. P225F mutants were resistant in vitro to all SDHIs tested. Similarly, isolates possessing the H272L mutation were highly resistant to boscalid but showed low to moderate levels of resistance to other SDHIs. The N230I mutants were moderately resistant to boscalid, fluopyram, and fluxapyroxad and showed low resistance levels to isopyrazam, bixafen, fenfuram, benodanil, and carboxin. The H272R mutants showed moderate levels of resistance to boscalid and low resistance levels to isopyrazam, fenfuram, and carboxin but remained sensitive to fluopyram, bixafen, fluxapyroxad, and benodanil. Similarly, the H272Y showed moderate levels of resistance to boscalid and very low resistance levels to isopyrazam, bixafen, fenfuram, and carboxin but showed increased sensitivity to benodanil and fluopyram. Boscalid provided moderate to high control of H272R/Y and N230I mutants in detached fruit assays but provided little control against the H272L and P225F mutants. In contrast, fluopyram controlled H272R/Y mutants and provided moderate levels of control toward H272L, N230I, and P225F mutants. Our findings suggest that sensitivity to SDHIs may vary greatly, dependent on the point mutation in the sdhb subunit.

Evaluation of plant-growth-promoting rhizobacteria, acibenzolar-S-methyl and hymexazol for integrated control of Fusarium crown and root rot on tomato.

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) can be potential agents for biological control of plant pathogens, while their combined use with conventional pesticides may increase their efficacy and broaden the disease control spectrum. The effect of four different Bacillus sp. PGPR strains (B. subtilis GB03 and FZB24, B. amyloliquefaciens IN937a and B. pumilus SE34) applied individually and in mixtures, as well as in combined use with acibezolar-S-methyl (ASM) and hymexazol, on plant growth promotion and on the control of Fusarium crown and root rot (FCRR) of tomato was evaluated. RESULTS: All PGPR strains promoted the tested plant growth characteristics significantly. A higher promoting effect was provided by SE34. Experiments on population dynamics of PGPR strains revealed that, after 28 days of incubation, populations of strain SE34 remained stable, while the remaining bacterial strains showed a slight decline in their population densities. The GB03 and FZB24 strains provided a higher disease suppression when applied individually. However, application of IN937a in a mixture with GB03 provided a higher control efficacy of Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Treatment of tomato plants with ASM resulted in a small reduction in disease index, while application of hymexazol provided significantly higher control efficacy. Combined applications of the four PGPR strains with either ASM or hymexazol were significantly more effective. CONCLUSION: The results of the study indicate that, when bacilli PGPR strains were combined with pesticides, there was an increased suppression of Forl on tomato plants, and thus they may prove to be important components in FCRR integrated management.

Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity.

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs) constitute a fungicide class with increasing relevance in crop protection. These fungicides could play a crucial role in successful management of grey mould disease. In the present study the effect of fluopyram, a novel SDHI fungicide, on several developmental stages of Botrytis cinerea was determined in vitro, and the protective and curative activity against the pathogen was determined on strawberry fruit. Furthermore, fungal baseline sensitivity was determined in a set of 192 pathogen isolates. RESULTS: Inhibition of germ tube elongation was found to be the most sensitive growth stage affected by fluopyram, while mycelial growth was found to be the least sensitive growth stage. Fluopyram provided excellent protective activity against B. cinerea when applied at 100 µg mL(-1) 96, 48 or 24 h before the artificial inoculation of the strawberry fruit. Similarly, fluopyram showed a high curative activity when it was applied at 100 µg mL(-1) 24 h post-inoculation, but, when applications were conducted 48 or 96 h post-inoculation, disease control efficacy was modest or low. The measurement of baseline sensitivity showed that it was unimodal in all the populations tested. The individual EC(50) values for fluopyram ranged from 0.03 to 0.29 µg mL(-1). In addition, no correlation was found between sensitivity to fluopyram and sensitivity to other fungicides, including cyprodinil, fenhexamid, fludioxonil, iprodione, boscalid and pyraclostrobin. CONCLUSIONS: The obtained biological activity, baseline sensitivity and cross-resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes.

Differences in Frequency of Transposable Elements Presence in Botrytis cinerea Populations from Several Hosts in Greece.

This study was conducted primarily to investigate the presence and frequency distribution of the transposable elements Boty and Flipper in populations of the necrotroph plant pathogen Botrytis cinerea in Greece. In total, 334 isolates were collected from diseased grape, strawberry, tomato, cucumber, kiwifruit, and apple fruit during 2009. The presence of the two transposable elements was based on polymerase chain reaction detection. Results showed that all the sampled hosts occurred in sympatry, with four possible different genotypes (transposa type carrying both transposable elements, Boty type carrying only the Boty element, Flipper type carrying only the Flipper element, and vacuma type carrying neither transposable element). Marked differences in genotype frequencies among populations were observed. In tomato, cucumber, grape, and strawberry, transposa isolates carrying both elements were predominant in the populations whereas, in kiwifruit and apple fruit populations, the vacuma isolates were prevailing. Furthermore, in kiwi and apple fruit populations, high frequencies of Flipper-type isolates were observed. In an attempt to explain the observed predominance of vacuma isolates in kiwifruit populations, the mycelial growth rate of a set of vacuma isolates was compared with the mycelial growth rate of a set of transposa isolates at three different temperatures (0, 10, and 20°C). The same set of isolates was used to compare pathogenicity of isolates on wound-inoculated kiwifruit incubated at two different temperatures (0 and 20°C), in terms of disease incidence and disease severity. In addition, the selected isolates were used to compare their ability in causing latent infections on kiwifruit in the field. The results showed that vacuma and transposa isolates had similar mycelial growth rates at the limiting temperatures of 0 and 10°C, while vacuma isolates grew faster at the optimum temperature of 20°C. Similarly, there was no significant difference regarding pathogenicity on kiwifruit between transposa and vacuma isolates. However, artificial inoculations conducted on blossoms in the field showed that vacuma isolates caused significantly higher incidence of latent infections.