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It is well-known that polyphenolic compounds are found abundantly in fruit, but various kinds of diseases lower these levels. This work measures total polyphenolic content, antioxidant activity and the levels of specific important antioxidants in fruits of the genetically modified (GM) plum variety HoneySweet, trees which were previously inoculated with a range of different virus infections. These were the Plum Pox virus (PPV), Prune Dwarf virus (PDV) and Apple Chlorotic Leaf-Spot virus (ACLSV). Uninoculated trees were used as controls. Antioxidant activity was measured using four different photometric methods – DPPH (2,2-diphenyl-1-picrylhydrazyl), DMPD (N-dimethyl-p-phenylenediamine), ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) and FRAP (Ferric reducing antioxidant power). Total polyphenol content was measured using the Folin–Ciocalteau method. The profiles of 10 specific antioxidant constituents in the fruits of the GM plum variety HoneySweet were detected and analyzed, since these are of interest for their role in human diets and could play a role in the resistance of plants to viruses. Detection was made using HPLC with UV-VIS detection. They were: gallic acid, p-coumaric acid, 4-aminobenzoic acid, chlorogenic acid, caffeic acid, ferulic acid, vanillin, rutin and quercetin. The compound with the highest concentration was chlorogenic acid (587 mg/100 g), and that with the lowest was p-coumaric acid (0.95 mg/100 g). Of the four methods of antioxidant activity used, in three the lowest levels of antioxidant activity were seen where the PPV virus was combined with ACLSV, and in three the highest levels were seen in the un-inoculated control without any infection. The highest values of total polyphenols were seen in the control (65.3 mg/100 g), followed by infection of PPV, then treatment PPV, PDV and ACLSV, then treatment PPV and PDV and finally the lowest levels were seen in treatment PPV and ACLSV (44.2 mg/100 g), which was also that with the lowest antioxidant activity.

The early stages of the fatigue damage in f.c.c. and b.c.c. metals are studied using high resolution techniques. The localization of the cyclic plastic strain results in formation persistent slip bands (PSBs) with specific dislocation structure. Characteristic surface relief is formed at locations where PSBs emerge on the surface. It consists of extrusions and intrusions separated by the original flat surface. Atomic force microscopy is used to study the details of the surface relief. The experimental findings are discussed in terms of the point defect model of fatigue crack initiation.

Background: The balance between antioxidants and pro-oxidants plays a significant role in the context of oxidative stress, influenced by both physiological and non-physiological factors. Objectives: In this study, 18 prescribed antibiotics (including doxycycline hydrochloride, tigecycline, rifampicin, tebipenem, cefuroxime, cefixime, potassium clavulanate, colistin, ampicillin, amoxicillin, amikacin, nalidixic acid, azithromycin, pipemidic acid trihydrate, pivmecillinam, aztreonam, fosfomycin sodium, and ciprofloxacin) were subjected to simultaneous determination of antioxidant and pro-oxidant potential to assess if pro-oxidant activity is a dominant co-mechanism of antibacterial activity or if any antibiotic exhibits a balanced effect. Methods: This study presents a recently developed approach for the simultaneous assessment of antioxidant and pro-oxidant potential on a single microplate in situ, applied to prescribed antibiotics. Results: Ten antibiotics from eighteen showed lower antioxidant or pro-oxidant potential, while five exhibited only mild potential with DPPH50 values over 0.5 mM. The pro-oxidant antioxidant balance index (PABI) was also calculated to determine whether antioxidant or pro-oxidant activity was dominant for each antibiotic. Surprisingly, three antibiotics—doxycycline hydrochloride, tigecycline, and rifampicin—showed significant measures of both antioxidant and pro-oxidant activities. Especially notable was tebipenem, a broad-spectrum, orally administered carbapenem, showed a positive PABI index ratio, indicating a dominant antioxidant over pro-oxidant effect. Conclusions: These findings could be significant for both therapy, where the antibacterial effect is enhanced by radical scavenging activity, and biotechnology, where substantial pro-oxidant activity might limit microbial viability in cultures and consequently affect yield.

Plum pox virus (PPV) is of the most important and harmful virus disease of plums, apricots, and peaches. Previous research has shown the non-transmissibility of PPV by aphids to genetically modified ’HoneySweet’ plum cultivar and to PK clone of BN4Kr myrobalan rootstock. In our experiments, the hypothesis was verified that non-transmissibility of PPV by aphids also exists in more resistant non-transgenic cultivars of plum or apricot. Therefore, relatively resistant cultivars of plum ’Gabrovská’, ’Anna Späth’, ’Althanova renklóda’ and apricot ’Harcot’, ’Hargrand’, ’Krajová raná’, grafted on the aphid transmission resistant myrobalan rootstock PK, were tested in pilot plantation conditions. None of the tested plum and apricot cultivars were naturally infected with PPV by aphid transmission. However, during monitoring in particular years, the successive die back of the ’Krajová’ and ’Hargrand’ apricot trees was noted, probably due to the poor affinity with PK myrobalan. Monitoring will continue in the following years.

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. ‘HoneySweet’ was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. ‘HoneySweet’ has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in ‘HoneySweet,’ the nature and stability of its sRNA profile, and the potential health risks of consuming ‘HoneySweet’ plums. Graft-challenged ‘HoneySweet’ trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.

A field trial with plum cv. HoneySweet was established in 2003 in an experimental plot near Crop Research Institute in Prague-Ruzyně, Czech Republic, on the basis of a permission issued by the Ministry of the Environment of the Czech Republic. In addition to the evaluation of resistance of plum cv. HoneySweet to Plum pox virus, the field trial was used to evaluate gene flow of the inserted transgene. Sampling of blackthorn and myrobalan trees outside the field test site occurred at distances ranging from 544 m to 845 m from the test site and showed no gene flow, testing both plants and seeds collected from blackthorns and myrobalans. Similarly, seeds from plums cv. Jojo growing directly at the field test place did not show any presence of the transgene after seven years of evaluation.

Resistance to Plum pox virus (PPV) in transgenic Prunus domestica L., clone C5 (cv. HoneySweet) was evaluated in a regulated field in the Czech Republic for fifteen years (2002-2016). PPV mild symptoms appeared in C5 trees only in several leaves situated close to the point of inoculum grafting up to 2010. No symptoms of PPV were observed in the years 2011-2013 and results of ELISA and RT-PCR detection tests were negative. In the twelfth year (2013), there was a severe unusual natural attack of plum trees by Monilinia sp. This Monilinia sp. attack occurred only one time - in 2013. There was no Monilinia sp. infection in 2002-2012 and in 2014-2016. Mild PPV symptoms reappeared in several leaves of transgenic plum trees in the next two years (2014-2015) and the presence of PPV was proved by DAS-ELISA and confirmed by RT-PCR.

Abstract  Apple cultivars ‘Idared’ and ‘Sampion’ were selected for application of in vitro thermotherapy at 39 ± 0.5°C. The presence of viruses in selected initial trees was detected by enzyme-linked immuno-sorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) before the beginning of thermotherapy. Cultivars ‘Idared’ and ‘Sampion’ were both infected with Apple chlorotic leaf spot virus (ACLSV) and with Apple stem pitting virus (ASPV). Cultivar ‘Sampion’ was moreover infected with Apple stem grooving virus (ASGV). Both apple cultivars were successfully multiplied in in vitro cultures on Murashige and Skoog (MS) medium with 1.5 mg l−1 6-benzylaminopurine (BAP). After the end of thermotherapy and transfer to ex vitro conditions, three clones of apple cultivar ‘Idared’ were free of both viruses (ACLSV, ASPV) according to repeated RT-PCR and ELISA. On the contrary, the same viruses (ACLSV, ASGV and ASPV) as in the initial field-grown trees were detected by RT-PCR in all four clones of cultivar ‘Sampion’. Other sanitation and testing of apple cultivars will continue in the following years. [ABSTRACT FROM AUTHOR]

Economically important viruses infect plums and other stone fruits cause lower yields, fruit size and quality and also affect its chemical composition. Fruits of the genetically modified (GM) plum 'HoneySweet' growing on trees deliberately infected with Plum pox virus (PPV), Prune dwarf virus (PDV) and Apple chlorotic leaf-spot virus (ACLSV) were analysed in detail to determine what effects these virus infections have on selected pomological traits, dissolved solids and titratable acids. Assessments of the fruits were made in the years from 2011 to 2014. The GM plum 'HoneySweet' was chosen for this experiment, and uninfected trees of the same variety in the same orchard were used as control. It was shown that there was no effect on dissolved solids resulting from PPV infections, either alone or in combination with the other viruses, but that there was a significant effect on levels of titratable acids where trees had been inoculated with all three viruses (treatment I). Regarding pomological traits, in most cases there were no significant effects seen to affect internal characteristics, only that in the assessments were lower although still of an acceptable quality for commercial fruit.

Sharka caused by Plum pox virus (PPV) is the most harmful disease of stone fruits in Europe and elsewhere in the world. There is no highly PPV resistant cultivar of plum. Biotech approach has led to the development of resistance through genetic engineering. In this study, we evaluated a transgenic plum Prunus domestica L., clone C5 (cv. HoneySweet), where the PPV resistance is based on RNA interference (RNAi). Resistance in C5 plums has been evaluated for PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV) in a regulated field trial in the Czech Republic for 12 years (2002–2013). Even under high and permanent infection pressure introduced through graft inoculation of the viruses, PPV has been detected in C5 trees only in several leaves situated close to the point of inoculum grafting in the first nine years. Mild symptoms of PPV disappeared year by year. No PPV symptoms were observed in the last three years and results of ELISA detection were negative. Similar results were obtained, when RT-PCR was used for PPV detection. No natural infection of PPV by aphids was recorded in C5 plums, even if PPV infected plants were growing in the same experimental field in close vicinity. Co-infections of PPV with PDV and/or ACLSV had practically no influence on stability of resistance in C5 trees. Twelve years of field testing in the Czech Republic clearly demonstrated the high level of resistance of C5 either to PPV infection by graft inoculation or natural infection through aphid vectors.

In this work two approaches to the description of short fatigue crack growth rate under large-scale yielding condition were comprehensively tested: (i) plastic component of the J-integral and (ii) Polák model of crack propagation. The ability to predict residual fatigue life of bodies with short initial cracks was studied for stainless steels Sanicro 25 and 304L. Despite their coarse microstructure and very different cyclic stress–strain response, the employed continuum mechanics models were found to give satisfactory results. Finite element modeling was used to determine the J-integrals and to simulate the evolution of crack front shapes, which corresponded to the real cracks observed on the fracture surfaces of the specimens. Residual fatigue lives estimated by these models were in good agreement with the number of cycles to failure of individual test specimens strained at various total strain amplitudes. Moreover, the crack growth rates of both investigated materials fell onto the same curve that was previously obtained for other steels with different properties. Such a “master curve” was achieved using the plastic part of J-integral and it has the potential of being an advantageous tool to model the fatigue crack propagation under large-scale yielding regime without a need of any additional experimental data.

Nine different visual evaluations of Plum pox virus (PPV) presence were carried out in four nurseries during 2012-2015. Results of visual evaluation were verified by ELISA. The presence of PPV was confirmed by ELISA in all the trees showing PPV symptoms. PPV has never been detected by ELISA in trees without PPV symptoms on leaves. A very low occurrence of PPV was proved in plums in all the four nurseries. There were two cases of higher occurrence of PPV in plum cultivars, where PPV originated from infected grafts. The PPV occurrence was 0.126 and 1.59%. The average occurrence of PPV in plums was 0.075%, 93 trees out of 123 630 inspected were PPV-infected. All the PPV-infected trees were destroyed immediately. No new PPV infection was proved later in season (August-September). In comparison with the situation in the 60s of the last century, the PPV occurrence in plums was 2.48% in one nursery in 1963 and there were nurseries with PPV occurrence exceeding 5%. All growing plants were destroyed in this case. The occurrence of PPV in the Czech fruit nurseries today is more than hundred times lower in comparison with the situation fifty years ago. The incidental occurrence of PPV in nurseries cannot be excluded in countries and areas with the endemic presence of PPV (in the Czech Republic and in most European countries). Visual inspection of PPV symptoms in nurseries confirmed by ELISA testing is sufficient. Infected plants must be removed immediately.

Sharka disease caused by the infection with the Plum pox virus (PPV) in stone fruit trees is worldwide the most devastating for stone fruit production. Until now, good sources of resistance to PPV within the peach group have not been available. There are no commercial cultivars of peach that are resistant to PPV. Other Prunus species are known to show varying levels of resistance. Interspecific hybrids GF 677 (Prunus amygdalus × P. persica) and Cadaman (P. davidiana × P. persica) were revealed to be resistant to PPV. The resistance to a Dideron isolate of the descendants of Cresthaven × GF 677 and Cresthaven × Cadaman and their progenitors was evaluated after inoculation by chip-budding in a sealed screenhouse. Results demonstrate a certain level of resistance in both progenies of interspecific hybrids and indicate a potential for PPV resistance transfer to commercial peach cultivars but it will be necessary to perform backcrosses with peach cultivars of agricultural interest in order to return pomological and agronomic traits. For the definitive confirmation of resistance/susceptibility it will be necessary to wait until the adult stage of hybrids.

Ten-year results of the practical immunity investigation of apricot cv. Harlayne are presented. Two-year-old trees of cv. Harlayne were inoculated by chip-budding with six different strains and isolates of Plum pox virus (PPV). PPV inoculated trees grew in the field and were evaluated from 2001 to 2011. No PPV symptoms appeared in the leaves of cv. Harlayne within ten years (2002-2011), and within eight years (2004-2011) in the fruits and stones. None of the six isolates of three different PPV strains was detected in the leaves and fruits by ELISA. Suckers of the rootstock Prunus myrobalana developed around cv. Harlayne trees in 2005-2011 were symptomless and ELISA was negative within seven years. New trees of cv. Harlayne obtained from tested trees by budding on PPV susceptible apricot rootstock MVA-2 in 2007 were PPV free from 2008 through 2011. The presence of PPV was proved by ELISA neither in leaves of cv. Harlayne nor in rootstock MVA-2.

Interspecific hybrids of Prunus persica, Barier, Fire, Cadaman, GF-677, and Prunus sp. hybrids and selections, MRS, NBS 540-73, and Pumiselect were evaluated for resistance to Plum pox virus. Hybrids were grafted onto trees of a peach cultivar artificially infected with PPV and evaluated for six years for resistance to the virus. The relative concentration of PPV protein was determined by semiquantitative ELISA in June every year. The presence of PPV in peach hybrids was confirmed by IC-RT-PCR in 2007-2008. The presence and intensity of PPV symptoms were evaluated monthly from May to September. The hybrid GF-677 (P. amygdalus × P. persica) was confirmed as highly resistant to PPV. Hybrids Cadaman (P. davidiana × P. persica) and Fire (P. amygdalus × P. persica) were characterized as resistant to PPV. Hybrids GF-677, Cadaman and Fire were selected as candidate sources of resistance to be crossed with peach cultivars susceptible to PPV.

The distribution of Plum pox virus (PPV) strains, PPV-D, PPV-M and PPV-Rec, was investigated in the Czech Republic in 2005-2008. Fifty-two to ninety-four samples of flowers or leaves of plum, myrobalan and blackthorn trees from different regions were tested in individual years. The presence of PPV was detected by DAS-ELISA with serotype-specific polyclonal antibodies. PPV-M was proved by DASI-ELISA with serotype-specific monoclonal antibodies; PPV-D, PPV-M and PPV-Rec were detected by RT-PCR in leaf samples from PPV infected trees. The presence of PPV-D ranged from 94.6% to 100%, the presence of PPV-M from 0.0% to 2.3% and the presence of PPV-Rec from 0.0% to 3.1% during 2005-2008. More than 95% of analysed samples of PPV were infected with PPV-D and less than 2.5% of analysed samples of PPV were infected with PPV-M or PPV-Rec. The presence of PPV-C was not proved in sweet cherry and sour cherry trees. The presence of PPV-EA was not proved in apricot trees.

Electron microscopy was used to detect the presence of virus particles or inclusions in growth tips and parenchymatic cells of leaves of plum, apricot and peach trees artificially infected with Plum pox virus (PPV). Typical pinwheels were found in ultrathin sections of leaves of PPV infected plums, apricots and peaches. Filamentous particles or their aggregates approximately 750 nm long were found in ultrathin sections of growth tips of plum, apricot, and peach shoots with a diameter of 0.5 mm. Pinwheels were never present in embryonic cells. No virus particles were found in ultrathin sections of growth tips of PPV infected plum, apricot and peach shoots of 0.2 mm in diameter. Embryonic cells of growth tips up to 0.2 mm in diameter are PPV free. PPV particles are present in growth tips at a distance 0.2-0.5 mm from the top; the virus is probably multiplied in this part of the growth tips.

Transgenic plums, Prunus domestica L. clone C5, were inoculated by bud grafting with Plum pox virus (PPV-Rec, recombinant strain originated from plum), PPV-Rec + Apple chlorotic leafspot virus (ACLSV), PPV-Rec + Prune dwarf virus (PDV), and PPV-Rec + ACLSV + PDV. Non-inoculated transgenic plums served as controls. Plants were grown in an open field for 5 years. They were evaluated by visible symptoms, by DAS-ELISA and RT-PCR. Mild PPV symptoms, diffuse spots or rings appeared two years after inoculation in some leaves of plants artificially inoculated with PPV-Rec, PPV-Rec + ACLSV, PPV-Rec + PDV, and PPV-Rec + ACLSV + PDV. Severe PPV symptoms appeared in leaves of shoots growing from infected buds used for inoculation. During the following three years, further weakening of PPV symptoms was observed in transgenic plants. In 2007, very mild PPV symptoms were found in only a few leaves, and over 60%, resp. 70% of the C5 trees showed no PPV symptoms. The presence of PPV was confirmed by ELISA, ISEM and RT-PCR. No difference in PPV symptoms was observed between PPV-Rec and combinations PPV-Rec + ACLSV, PPV-Rec + PDV, PPV-Rec + ACLSV + PDV. No symptoms of ACLSV appeared in combinations of ACLSV with PPV-Rec and PPV-Rec + PDV during 2004-2007, but the presence of ACLSV in leaves of transgenic plants clone C5 was proved by ELISA and RT-PCR. Neither synergistic nor antagonistic effects of ACLSV on PPV-Rec were observed. No symptoms of PDV appeared in combinations of viruses with PDV during 2004-2007. PDV was not detected by ELISA, and the presence of PDV was uncertain by RT-PCR in most of inoculated trees in 2006 and 2007. The results of RT-PCR will be further confirmed by sequence analysis and discussed. These results suggest a possible antagonistic interaction between PPV-Rec and PDV in plum clone C5.

The distribution of Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic ringspot virus (ACLSV) and Apple mosaic virus (ApMV) in naturally growing shrubs of blackthorn and road-bordering trees of plum and myrobalan, and of PPV, PDV, PNRSV and Cherry leafroll virus (CLRV) in sweet and sour cherry trees were investigated. The most widely distributed viruses were PPV in plums (74% of the investigated trees were infected); PPV, PDV, and PNRSV in myrobalans (26%, 11% and 18%, respectively), PDV in blackthorns (27%), and PDV and PNRSV in cherries (25% and 22%). PPV was not detected in sweet and sour cherries. The incidence of ACLSV and ApMV was negligible in individually growing trees of the genus Prunus in the Czech Republic.