Ivana Šola

Separation of hypoviral double-stranded RNA on monolithic chromatographic supports

A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.

Physcomitrella patens auxin conjugate synthetase (GH3) double knockout mutants are more resistant to Pythium infection than wild type

Auxin homeostasis is involved in many different plant developmental and stress responses. The auxin amino acid conjugate synthetases belonging to the GH3 family play major roles in the regulation of free indole-3-acetic acid (IAA) levels and the moss Physcomitrella patens has two GH3 genes in its genome. A role for IAA in several angiosperm--pathogen interactions was reported, however, in a moss--oomycete pathosystem it had not been published so far. Using GH3 double knockout lines we have investigated the role of auxin homeostasis during the infection of P. patens with the two oomycete species, Pythium debaryanum and Pythium irregulare. We show that infection with P. debaryanum caused stronger disease symptoms than with P. irregulare. Also, P. patens lines harboring fusion constructs of an auxin-inducible promoter from soybean (GmGH3) with a reporter (ß-glucuronidase) showed higher promoter induction after P. debaryanum infection than after P. irregulare, indicating a differential induction of the auxin response. Free IAA was induced upon P. debaryanum infection in wild type by 1.6-fold and in two GH3 double knockout (GH3-doKO) mutants by 4- to 5-fold. All GH3-doKO lines showed a reduced disease symptom progression compared to wild type. Since P. debaryanum can be inhibited in growth on medium containing IAA, these data might indicate that endogenous high auxin levels in P. patens GH3-doKO mutants lead to higher resistance against the oomycete.

Analysis of salicylic acid‐dependent pathways in Arabidopsis thaliana following infection with Plasmodiophora brassicae and the influence of salicylic acid on disease

Salicylic acid (SA) biosynthesis, the expression of SA-related genes and the effect of SA on the Arabidopsis-Plasmodiophora brassicae interaction were examined. Biochemical analyses revealed that, in P. brassicae-infected Arabidopsis, the majority of SA is synthesized from chorismate. Real-time monitored expression of a gene for isochorismate synthase was induced on infection. SA can be modified after accumulation, either by methylation, improving its mobility, or by glycosylation, as one possible reaction for inactivation. Quantitative reverse transcription-polymerase chain reaction (qPCR) confirmed the induction of an SA methyltransferase gene, whereas SA glucosyltransferase expression was not changed after infection. Col-0 wild-type (wt) did not provide a visible phenotypic resistance response, whereas the Arabidopsis mutant dnd1, which constitutively activates the immune system, showed reduced gall scores. As dnd1 showed control of the pathogen, exogenous SA was applied to Arabidopsis in order to test whether it could suppress clubroot. In wt, sid2 (SA biosynthesis), NahG (SA-deficient) and npr1 (SA signalling-impaired) mutants, SA treatment did not alter the gall score, but positively affected the shoot weight. This suggests that SA alone is not sufficient for Arabidopsis resistance against P. brassicae. Semi-quantitative PCR revealed that wt, cpr1, dnd1 and sid2 showed elevated PR-1 expression on P. brassicae and SA + P. brassicae inoculation at 2 and 3 weeks post-inoculation (wpi), whereas NahG and npr1 showed no expression. This work contributes to the understanding of SA involvement in the Arabidopsis-P. brassicae interaction.

Involvement of kaempferol in the defence response of virus infected Arabidopsis thaliana

The roles of several phenolic compounds in plant defence response have been extensively studied, yet little is known about the role of flavonoids in plant-virus interaction. Quantitative and qualitative changes of selected phenolics in Arabidopsis thaliana induced by Cucumber mosaic virus containing satellite RNA (CMVsat) infection were analysed accompanied by plant hormone, chalcone synthase and pathogenesis-related gene expression analysis. Lower leaves of infected plants had a lower concentration of total phenolics compared to control plants. The concentration of kaempferol in upper leaves of all infected plants was significantly lower compared to control plants, while the expression of the chalcone synthase gene in those leaves was in most cases upregulated. All infected plants had a higher concentration of indole-3-acetic acid in lower leaves, which was accompanied with a lower concentration of kaempferol in upper leaves. Our research demonstrates a correlation between kaempferol and indole-3-acetic acid in response to CMVsat infection in Arabidopsis. We demonstrated two different metabolic patterns in infected plants suggesting the activation of two different defence responses. We also propose kaempferol to be an important part of the auxin-dependent defence response which limits systemic movement of CMVsat and that this defence response is activated prior to the well-known salicylic acid dependent defence response. Further research on kaempferol and its role in Arabidopsis-CMVsat interaction will improve our understanding on the role of flavonoids in plant defence.

The effect of biostimulant and fertilizer on “low input” lettuce production

Abstract Lettuce production in the winter on the Adriatic coast, especially in a non-heated plastic tunnel, requires longer cultivation and is characterised by lower head mass and yield. In these conditions, the effect of biostimulant Bio-algeen S-90 and fertilizer Megagreen on the production of the traditional winter lettuce cultivar ‘Four Seasons’ was tested. Both treatments showed a positive effect on the growth and total yield of winter lettuce, and decreased the share of non-marketable yield. Bio-algeen S-90 treatment increased the plant height by 61.5%, and foliar treatment with Megagreen by 60.9%, as compared to the control treatment. Equally, both treatments resulted in higher leaf numbers (47.7% for Bio-algeen S-90 and 37.2% for Megagreen). The head mass of lettuce treated with Bio-algeen S-90 and Megagreen was 30.3% and 25.0% higher than in the control treatment, respectively. Megagreen contributed more to chlorophyll and carotenoid content, while Bio-algeen S-90 elevated the amount of vitamin C and dry matter. The pH value of lettuce juice decreased after Bio-algeen S-90, while the mineral content (N, P and K) did not differ between the tested treatments. Lower nitrate content was detected after both treatments. The obtained results elucidate the effect of Bio-algeen S-90 and Megagreen on “low input” lettuce production.

Europium improves the transport of quercetin through Arabidopsis thaliana

The effect of a rare earth element europium (Eu) on the long-distance transport of a plant defence compound quercetin (Q) was investigated. The complex Q/Eu3+ was synthesized in a HEPES buffer and tested for its transport ability through Arabidopsis thaliana and its ability to interact with target molecules in plant cells. Our results show that complexation with Eu3+ enhanced the transport of Q through Arabidopsis roots. During the transport, the complex degraded and released a free Q to tissues where Q was originally not available. Thus, the plant became better supplied with the defensive compound Q. The obtained spectrophotometric data imply that one of the reasons for the Q/Eu3+ degradation was the interaction of the complex with double stranded RNAs (dsRNAs) present in Arabidopsis. Since dsRNAs are replicative forms of plant RNA viruses, the ability of Q/Eu3+ to release a free Q in their presence suggests further investigation of this complex as a potential antiviral agent.

Effects of biodynamic production on growth and essential oil content in basil

Abstract The effects of a biodynamic sowing calendar on the growth (plant height, fresh herb yield, nodes number) and quality (percentage of leaf mass, essential oil content) of three basil species, Ocimum americanum L., Ocimum × hybrida and Ocimum basilicum L., represented by the cultivars ‘Rosso’ and ‘Eco Genovese’, were tested. Statistical analyses showed that the species had greater impact on the observed parameters than either the sowing date or the species and sowing date in combination. The species showed a significant influence on all five tested parameters, while sowing date and interaction of both factors significantly influenced plant height, leaf percentage and essential oil content. The best yield per plant was obtained for O. × hybrida and O. basilicum ‘Eco Genovese’. The lowest species O. × hybrida produced the highest amount of essential oil. »Nodes number« parameter most clearly separated the species, but not the cultivars. Even though O. americanum gave the tallest plants, it did not yield either the highest amount of fresh herb or essential oil. This species’ height was most consistent, considering the significant impact of biodynamic rhythm. Sowing date was not crucial for basil fresh yield; however if there is a need for taller plants with a higher percentage of leaf mass and more essential oil, sowing date needs to be controlled.