István Tóbiás

Immunoassays for plant cytokinins as tools for the assessment of environmental stress and disease resistance.

The level of cytokinin type hormones present in plant tissues, such as N 6 -(2-isopentenyl)-adenosine (IPA), N 6 -(2isopentenyl)-adenosine (2-iP), trans-zeatin riboside (ZR) and trans-zeatin (Z) is a good indicator of the resistance of plants to abiotic environmental stresses and to necrotic pathogens. Hapten-homologous and hapten-heterologous competitive indirect enzyme-linked immunosorbent assays (ELISAs) were developed, allowing the use of minute amounts of plant extracts for cytokinin analysis. These assays were used for the detection of members of the cytokinin plant hormone family including IPA and ZR types. The assays, in optimized formats, readily detected these plant hormones at concentration levels of 2‐5 ng ml 1 , and showed high specificity for selected cytokinins. Certain assay parameters (e.g. the type of tracer enzyme, incubation and preincubation time, etc.) had a strong influence on detection sensitivity. Nonetheless, the assays appear robust showing tolerance to pH and to several water-miscible organic solvents. The described ELISA systems were sensitive enough to detect endogenous hormone levels in crude plant extracts without intense purification. In vitro selected transgenic tobacco and tomato lines showing tolerance to several stress factors proved to have higher levels of cytokinins than the corresponding control plants indicating that the developed immunoassay is suitable for the determination of stress resistance of plants by monitoring their cytokinin content.

Replacement of the coat protein gene of plum pox potyvirus with that of zucchini yellow mosaic potyvirus: characterization of the hybrid potyvirus

Infectious hybrid virus was generated by replacing part of the coat protein gene of plum pox potyvirus with that of the zucchini yellow mosaic potyvirus. This viable hybrid contains 84.5% of zucchini yellow mosaic potyvirus coat protein gene while the rest of the sequence was derived from plum pox potyvirus. Changing the coat protein gene between these two viruses had no effect on the experimental host range. Pathogenicity, stability and replication capacity of the hybrid virus were nearly identical to the parent viruses.

Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions.

Phytohormone levels and the expression of genes encoding key enzymes participating in hormone biosynthetic pathways were investigated in pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation led to the development of hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a systemic, compatible interaction. ObPV-inoculation markedly increased not only the levels of salicylic acid (SA) (73-fold) and jasmonic acid (8-fold) but also those of abscisic acid, indole-3-acetic acid, indole-3-butyric acid, cis-zeatin, cis-zeatin-9-riboside and trans-zeatin-9-riboside in the inoculated pepper leaves 3 days post inoculation. PMMoV infection increased only the contents of gibberellic acid and SA. Hormone contents did not change significantly after ObPV or PMMoV infection in non-infected upper leaves 20 days post inoculation. Concentrations of some brassinosteroids (BRs) and progesterone increased both in ObPV- and PMMoV inoculated leaves. ObPV inoculation markedly induced the expression of three phenylalanine ammonia-lyase (PAL) and a 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes, while that of an isochorismate synthase (ICS) gene was not modified. PMMoV inoculation did not alter the expression of PAL and ICS genes but induced the transcript abundance of ACO although later than ObPV. Pre-treatment of pepper leaves with exogenous 24-epi-brassinolide (24-epi-BR) prior to ObPV-inoculation strongly mitigated the visible symptoms caused by ObPV. In addition, 24-epi-BR pre-treatment markedly altered the level of several hormones in pepper leaves following ObPV-inoculation. These data indicate that ObPV- and PMMoV-inoculations lead to intricate but well harmonized hormonal responses that are largely determined by the incompatible or compatible nature of plant-virus interactions.

Progesterone moderates damage in Arabidopsis thaliana caused by infection with Pseudomonas syringae or P. fluorescens

Brassinosteroids are known to protect plants against various abiotic and biotic stresses, however, very limited information is available about the role of progesterone. Therefore the effects of Pseudomonas syringae pv. syringae (P.s.) wild type strain 61, its hrcC mutant, and the saprophytic P. fluorescens (P.f.) strain 55 were investigated in wild type Arabidopsis thaliana cv. Columbia and its rbohF knock-out mutant, with and without progesterone pre-treatment. The reactions of wild type and rbohF mutant Arabidopsis to bacterial inoculations were similar, although 2 h after injection of P.s. a larger increase of electrolyte leakage was measured in wild type than in rbohF knockout mutant leaves. The hrcC mutant caused weak necrotic symptoms and increased leakage in both types of Arabidopsis, although to a much lesser extent than P.s. The P.f. did not induce any visible symptom, but slightly increased the electrolyte leakage in both types of Arabidopsis. Inoculation by all Pseudomonas bacteria led to significant alterations in photosystem 2 efficiency as compared to control plants. Pre-treatment of leaves with progesterone diminished the necrotic symptoms, the electrolyte leakage and improve the efficiency of photosystem 2 caused by Pseudomonas bacteria.

The nucleotide sequence of barley strain of Wheat dwarf virus isolated in Hungary.

Barley-infecting isolates of WDV were collected in the field of near Sofia. The complete genomes of two isolates were amplified by PCR, cloned into pGEM-T plasmid and sequenced. The two clones were the same size and showed complete homology. The WDV-Bg17 clone was compared with Barley dwarf virus, Oat dwarf virus and Wheat dwarf virus isolates. Based on DNA sequences WDV-Bg17 isolate shows high homology (95–97%) to Barley dwarf virus isolates and differs from Oat dwarf virus (71% homology) and Wheat dwarf virus (85% homology).

Characterization of a natural Plum pox virus isolate bearing a truncated coat protein

Plum pox virus (PPV) isolates were collected in Hungary from plum varieties. PCR targeting the 3′ genomic region resulted in a shorter PCR product in the case of the B1298 isolate bearing a 135-nucleotide deletion in frame in the N-terminal part of the coat protein (CP). The isolate was aphid-transmissible and the virion diameter was reduced compared to PPV-SK68. Detectability of this isolate by Western blot varied according to the antibody used. Integration of the deleted CP gene into an infectious PPV clone had no effect on infectivity and symptomatology. In competition experiments, B1298 had a considerable advantage in virus accumulation.

Phylogenetic analysis of Tomato spotted wilt virus (TSWV) NSs protein demonstrates the isolated emergence of resistance-breaking strains in pepper

Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010–2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

Comparative studies of compatible and incompatible pepper–Tobamovirus interactions and the evaluation of effects of 24-epibrassinolide

The aim of study was to gain a deeper knowledge about local and systemic changes in photosynthetic processes and sugar production of pepper infected by Obuda pepper virus (ObPV) and Pepper mild mottle virus (PMMoV). PSII efficiency, reflectance, and gas exchange were measured 48 and/or 72 h after inoculation (hpi). Sugar accumulation was checked 72 hpi and 20 d after inoculation (as a systemic response). Inoculation of leaves with ObPV led to appearance of hypersensitive necrotic lesions (incompatible interaction), while PMMoV caused no visible symptoms (compatible interaction). ObPV (but not PMMoV) lowered Fv/Fm (from 0.827 to 0.148 at 72 hpi). Net photosynthesis decreased in ObPV-infected leaves. In ObPV-inoculated leaves, the accumulation of glucose, fructose, and glucose-6-phosphate was accompanied with lowered sucrose, maltoheptose, nystose, and trehalose contents. PMMoV inoculation increased the contents of glucose, maltose, and raffinose in the inoculated leaves, while glucose-6-phosphate accummulated in upper leaves.

First record of the family Penthaleidae (Acari) in Hungary: Morphological and Molecular approaches of the Hungarian Penthaleus cf. major (Dugès, 1837)

The first Hungarian record of the family Penthaleidae, the occurrence of blue oat mite [Penthaleus cf. major (Duges, 1837)] in Hungary is presented. This pest mite species was collected on lettuce in greenhouse. Notes to the morphology and the three (cox 1, 18sRNA, ITS2) sequences of the Hungarian specimens are given.