Dariusz Załuski

Behavioral sensitivity of Myzus persicae to volatile isoprenoids in plant tissues

Volatile isoprenoids (VIPs) are low‐molecular isoprene‐derived compounds that are produced and emitted by plants as a protection against and in response to various biotic and abiotic stresses. They also participate in direct and indirect plant defense against herbivores. Foliar VIPs may accumulate in mesophyll cells, which makes them detectable for foraging aphids. In this study, probing behavior of the peach potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), was monitored using the electrical penetration graph (EPG) technique, following the exogenous application of camphene, α‐ and β‐ionones, and linalool to its host plant Brassica rapa subsp. pekinensis (Lour.) Hanelt (Brassicaceae). None of the VIPs studied affected M. persicae propensity to probe: aphids spent at least 75% of the experimental time penetrating plant tissues. The application of α‐ionone and linalool did not alter M. persicae behavior relative to control individuals. In contrast, on camphene‐ and β‐ionone‐treated plants, the proportion of non‐probing relative to other stylet activities was high, the success rate in reaching sieve elements and feeding was low, and the proportion of salivation in phloem phase was high. The aphid reluctance to continue probing and feeding due to the exogenously applied VIPs may be considered as means for the limitation of transmission of semi‐persistent and persistent viruses.

Development of an FgMito assay: A highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto

An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r(2)-values of >0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.

trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto

Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time.

Sinapic Acid Affects Phenolic and Trichothecene Profiles of F. culmorum and F. graminearum Sensu Stricto

Plant-derived compounds for reducing the mycotoxin load in food and feed have become a rapidly developing research field of importance for plant breeding efforts and in the search for natural fungicides. In this study, toxigenic strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to sinapic acid on solid YES media at levels close to those reported in wheat bran. Fusaria produced phenolic acids, whose accumulation was decreased by exogenous sinapic acid. Strains exposed to the lowest doses of sinapic acid showed more efficient reduction of phenolic acid production than fungi kept at higher concentrations of this compound. Fungi reduced exogenous sinapic acid, leading to the formation of syringic aldehyde. Treatment with sinapic acid led to a dramatic accumulation of its parent compound ferulic acid, presumably due to inhibition of the further conversion of this phenolic compound. Exogenous sinapic acid decreased the production of trichothecenes by fungi. Higher doses of sinapic acid resulted in more efficient reduction of mycotoxin accumulation in the media. Gene expression studies of Tri genes responsible for trichothecene biosynthesis (Tri4, Tri5 and Tri10) proved that the inhibition of mycotoxin production by sinapic acid occurred at the transcriptional level. Fusaria respond to sinapic acid by stimulation of ergosterol biosynthesis.

Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum

Fusarium culmorum is a ubiquitous, soil-borne fungus (ascomycete) causing foot and root rot and Fusarium head blight on cereals. It is responsible for yield and quality losses as well as grain contamination with mycotoxins, which are a potential health hazard. An extremely sensitive mitochondrial-based qPCR assay (FcMito qPCR) for quantification of F. culmorum was developed in this study. To provide specificity, the FcMito assay was successfully validated against 85 F. culmorum strains and 53 isolates of 30 other fungal species. The assay efficiency and sensitivity were evaluated against different F. culmorum strains with various amounts of pure fungal DNA and in the presence of background wheat DNA. The results demonstrated the high efficiency of the assay (97.2–106.0%, R2-values > 0.99). It was also shown that, in the presence of background DNA, 0.01 pg of fungal template could be reliably quantified. The FcMito assay was used to quantify F. culmorum DNA using 108 grain samples with different trichothecene levels. A significant positive correlation was found between fungal DNA quantity and the total trichothecene content. The obtained results showed that the sensitivity of the FcMito assay was much higher than the nuclear-based qPCR assay for F. culmorum.

Changes in Phenylpropanoid and Trichothecene Production by Fusarium culmorum and F. graminearum Sensu Stricto via Exposure to Flavonoids

Flavonoids are a group of hydroxylated polyphenolic compounds widely distributed in the plant kingdom. Biosynthesis of these compounds involves type III PKSs, whose presence has been recently predicted in some fungal species through genome sequencing efforts. In this study, for the first time it was found that Fusaria produce flavonoids on solid YES medium. Naringenin, as the central precursor of all flavonoids, was produced at highest quantities, followed by quercetin, kaempferol, apigenin and luteolin. In plants, flavonoids are involved in the protection of cereals to a wide range of stresses, including host defense against Fusaria. Under in vitro conditions, strains of Fusarium culmorum and F. graminearum sensu stricto were incubated at levels of flavonoids close to amounts produced by cereals in response to fungal infection. The amounts of exogenous naringenin, apigenin, luteolin, kaempferol and quercetin were reduced and converted by fungi to the other flavonoid derivatives. Treatment of fungi with naringenin derivatives led to the inhibition of naringenin production. Correspondingly, the production of fungal-derived phenolic acids decreased in flavonoid treated samples, although this effect appeared to be dependent on the strain, flavonoid molecule and its concentration. Fusaria showed high variability in trichothecene production in response to flavonoids. With emphasis on quercetin, mycotoxin accumulation in the media was significantly decreased by luteolin, kaempferol, naringenin and apigenin. However, in some cases, apigenin led to the increase of mycotoxin content in the media. Gene expression experiments of Tri genes responsible for trichothecene biosynthesis (Tri4, Tri5 and Tri10) proved that the inhibition of mycotoxin production by flavonoids occurred at the transcriptional level. However, the changes in Tri transcript levels were not significant in most apigenin and all kaempferol-treated cultures. In this study, a link was established between antioxidant and antiradical properties of flavonoids and their effects on fungi.

Evaluation of biomass quality of selected woody species depending on the soil enrichment practice

Abstract Perennial energy crops are a source of the bio-mass used to generate energy. The aim of this study was to determine the chemical and thermophysical parameters of short rotation woody crops (black locust, poplar and willow), depending on soil enrichment practice (mineral fertilisation, lignin and mycorrhiza), in three- and four-year harvest cycles. In the study, the thermophysical properties and elemental composition of the biomass were determined. All analyses were performed in trip-licate according to the standards. The fresh black locust biomass had the lowest moisture content, which resulted in the best lower heating value (10.16 MJ kg−1, on average) in the four-year harvest cycle. The poplar biomass had the greatest higher heating value, fixed carbon, carbon and ash content, the highest concentrations of which were found in the biomass in which lignin was applied (2.00% d.m.). On the other hand, the willow biomass contained the lowest concentrations of ash and fixed carbon. Soil enrichment significantly differentiated the quality parameters of black locust, poplar and willow. This effect is of particular importance to those who grow and use biomass as a fuel.