Multidrug resistance of Penicillium expansum to fungicides: whole transcriptome analysis of MDR strains reveals overexpression of efflux transporter genes.

Penicillium expansum is the most common apple fruit postharvest spoilage agent that causes a disease known as Blue Mold. Disease control is based on fungicide use. However, development of resistance to fungicides hampers the success of this control method. Fungicide sensitivity monitoring studies in Greece revealed the presence of pathogen strains exhibiting simultaneous resistance to different chemically unrelated compounds (multidrug resistance, MDR). This study was initiated aiming primarily to test the hypothesis that the MDR phenotype is associated with overexpression of efflux transporter genes and to determine the fitness of the MDR isolates. The monitoring study (n = 264) and the measurements of sensitivity in terms of EC50 values to 9 different compounds revealed that almost 5% of the population was of the MDR type. In the selected MDR isolates, the highest resistant factors were calculated for fludioxonil and pyraclostrobin, while the same isolates were moderately resistant to cyprodinil, thiophanate methyl and fluxapyroxad. In the resistant strains no target site mutations were detected in the target genes of each fungicide class, while in addition, a synergistic activity was observed between fungicides and the drug transporter modulator verapamil in some isolates. To obtain a direct insight on the resistance mechanism, the transcriptome of 2 MDR and 1 sensitive isolates was sequenced using Illumina HiSeq 2500 and differences in efflux transporter gene expression profile were figured out. Gene expression profiling analysis was performed before and after the exposure of fungal mycelia to fludioxonil. This analysis revealed the up-regulation of several MFS transporter genes and a limited number of ABC transporter genes either before or after the exposure to fludioxonil in the MDR isolates. Expression results for genes with the highest expression levels were verified by qRT-PCR assays. Fitness components measurements revealed that MDR isolates were of lower mycelial growth and pathogenicity compared to sensitive strains but they were producing higher number of conidia. The above mentioned data represent the first report of MDR in P. expansum associated with overexpression of drug efflux transporters and contribute to our knowledge in the mechanisms associated with fungicide resistance development in this fungal species.

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.

Effect of specific plant-growth-promoting rhizobacteria (PGPR) on growth and uptake of neonicotinoid insecticide thiamethoxam in corn (Zea mays L.) seedlings.

BACKGROUND: Corn (Zea mays L.) is one of the most important cereal crops in the world and is used for food, feed and energy. Inoculation with plant-growth-promoting rhizobacteria (PGPR) would reduce the use of chemical fertilisers and pesticides and could be suggested as an alternative practice for sustainable production of corn in modern agricultural systems. In this study, the effect of two Bacillus PGPR formulated products, Companion (B. subtilis GB03) and FZB24 (B. subtilis FZB24), on corn growth and root uptake of insecticide thiamethoxam was investigated. RESULTS: All bacterial treatments enhanced root biomass production by 38-65% compared with the uninoculated control, with no stimulatory effect of PGPR on above-ground biomass of corn. The uptake results revealed that, in plants inoculated with the PGPR B. subtilis FZB24 and B. subtilis GB03, singly or in combination, the uptake and/or systemic translocation of thiamethoxam in the above-ground corn parts was significantly higher at the different growth ages compared with the control receiving no bacterial treatment. CONCLUSION: The findings suggest that the PGPR-elicited enhanced uptake of thiamethoxam could lead to improved efficiency of thiamethoxam using reduced rates of pesticides in combination with PGPR as an alternative crop protection technique.

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.

Biodegradation of soil-applied pesticides by selected strains of plant growth-promoting rhizobacteria (PGPR) and their effects on bacterial growth.

A laboratory study was conducted to investigate the influence of four PGPR strains on the degradation of five soil applied pesticides and their effects on bacterial growth. Interactions of Bacillus subtilis GB03, Bacillus subtilis FZB24, Bacillus amyloliquefaciens IN937a and Bacillus pumilus SE34 with two concentrations of acibenzolar-S-methyl, metribuzin, napropamide, propamocarb hydrochloride and thiamethoxam in liquid culture and soil microcosm were studied. The degradation of acibenzolar-S-methyl by all PGPR tested in low and high concentration, was 5.4 and 5.7 times, respectively, faster than that in non-inoculated liquid culture medium. At the end of the 72-h liquid cultured experiments, 8-18, 9-11, 15-36 and 11-22% of metribuzin, napropamide, propamocarb hydrochloride and thiamethoxam, respectively, had disappeared from PGPR inoculated medium. Under the soil microcosm experimental conditions, the half-lives of acibenzolar-S-methyl incubated in the presence of PGPR strains spiked at 1.0 and 10.0 mg kg(-1) were 10.3-16.4 and 9.2-15.9 days, respectively, markedly lower compared with >34.2 days in the control. From the rest pesticides studied degradation of propamocarb hydrochloride and thiamethoxam was enhanced in the presence of B. amyloliquefaciens IN937a and B. pumilus SE34. Acibenzolar-S-methyl, propamocarb hydrochloride and thiamethoxam significantly increased the PGPR growth. However, the stimulatory effect was related to the level of pesticide spiked.

Determination of acibenzolar-S-methyl and its acidic metabolite in soils by HPLC-diode array detection.

A simple and accurate method for the analysis of acibenzolar-S-methyl (benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester; CGA 245 704; ASM) and its major conversion product, benzo[1,2,3]thiadiazole-7-carboxylic acid (CGA 210 007; BTC), in soils is presented. ASM extraction from soil samples was performed using acetonitrile and BTC was extracted with a mixture of potassium phosphate buffer (0.5 M, pH 3) and acetonitrile (70:30 %, v/v). Both extracts were directly analyzed in a high-performance liquid chromatography-diode array detection (HPLC-DAD) system. Pesticide separation was achieved on a C18 (4.6 mm × 150 mm, 5 µm) analytical column with a isocratic elution of acetonitrile:water 40:60 % (v/v) with 0.6 mL L⁻¹ acetic acid at a flow rate of 1 mL min⁻¹. Linear regression coefficients (r (2)) of the external calibration curves were always above 0.9997. The limits of detection (LOD) and quantification (LOQ) of the method were 0.005 and 0.02 mg kg⁻¹ for ASM, and 0.01 and 0.05 mg kg⁻¹ for BTC, respectively. Recoveries were investigated at six fortification levels and were in the range of 90-120 % for ASM and 74-96 % for BTC with relative standard deviations (RSDs) below 11 % in all cases. The method was also validated by analyzing freshly spiked soil samples with 2.7% organic matter content at 0.5 mg kg⁻¹ level, with slightly lower recovery values only for ASM. Moreover, recoveries for intermediate aged residues of the analytes were similar to fresh residues. This method was also applied to determine ASM half-life (t(½) = 8.7 h) and the rate of the acidic metabolite formation.