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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.

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.

Trees of prune (Prunus domestica L.), cv. Jojo, were inoculated by chip budding with three different strains of PPV isolated from European plum in the Czech Republic. These isolates included Plum pox virus M strain (PPV-M), Plum pox virus D strain (PPV-D) and a PPV-recombinant both strains (PPV-Rec). The results of the evaluation of the inoculated trees over 2 years are presented. Trees of plum cv. Jojo behaven differently to infection with the three PPV strains. A strong hypersensitive reaction appeared a year after inoculation with PPV-M and PPV-Rec strains, although not all inoculated tree died. PPV must have been present in the tissue of cv. Jojo because the virus was transferred to the rootstock St. Julien. Plants of the rootstock became systemically infected with the PPV-M and PPV-Rec strains, showing severe PPV symptoms. The presence of PPV was proved by ELISA in leaves of rootstock St. Julien, but not in leaves of cv. Jojo. Inoculation with strain PPV-D resulted in partial hypersensitive reaction of plants of cv. Jojo, but after initial stunting and partial death of shoots recovering of plants was observed.

Cherry leaf roll virus (CLRV), Myrobalan latent ringspot virus (MLRSV) and Strawberry latent ringspot virus (SLRSV) were transferred by budding to woody trees, hybrid Ishtara, peach cv. GF 305 and cv. Lesiberian. Three buffers with antioxidants and stabilisers: 0.01M phosphate with 1% caffeine; 0.007M phosphate-0.01M veronal with 0.01M cysteine hydrochloride and 0.007 EDTA; 0.015M phosphate with 1% nicotine and 0.066M phosphate buffer without additives were compared for their efficiency in mechanical transmission from woody sources to herbaceous hosts (Chenopodium quinoa and C. amaranticolor). 0.007M phosphate-0.01M veronal buffer with 0.01M cysteine hydrochloride, and 0.007 EDTA and 0.015M phosphate buffer with 1% nicotine were found to be the best buffers for the three nepoviruses. Both biological transmission to herbaceous assay hosts and detection by ELISA in the investigated tree are necessary to reliably detect the three nepoviruses. Biological detection is reliable from April to June, and in September and October. ELISA detection is also more difficult in July and August. The suitability of C. quinoa and C. amaranticolor to maintain CLRV, MLRSV and SLRSV was compared. C. amaranticolor plants were found to be more suitable for CLRV and SLRSV, infected plants grow over 6 months after mechanical inoculation by the nepoviruses. C. quinoa plants proved to be most suitable for maintenance of MLRSV, while C. amaranticolor is a symptomless host of MLRSV. Reinoculation with the nepoviruses is recommended in intervals of 4 to 6 months.

So far, the identification of the nematode species Xiphinema vuittenezi relied mainly on time-consuming morphological and morphometrical studies. Therefore, a polymerase chain reaction (PCR) protocol was optimised that both reliably and rapidly identifies X. vuittenezi. The internal transcribed spacer (ITS) species-specific primer of ribosomal DNA gene of X. vuittenezi was used. Nine populations of this species from Central Bohemia were investigated by means of PCR.

The suitability of different apple tissues for Apple stem grooving virus (ASGV) detection throughout the year was checked by RT-PCR and ELISA. Detectable amounts of ASGV were generally found in all tissues (bark, dormant buds, petals and leaves) tested by RT-PCR from January to mid-June. Leaves during flowering (in May) were the most suitable tissues for the virus detection by both methods (RT-PCR and ELISA). The leaves collected in summer (June, July and August) or other tissues such as bark, dormant buds and petals were not reliable for ASGV detection by ELISA.

Twenty-eight American peach cultivars were screened for their reaction to Plum pox virus (PPV). The cultivars were evaluated for the presence and intensity of PPV symptoms in leaves and fruits, and the relative concentration of PPV protein in flowers was determined by ELISA. The results allowed to divide the cultivars into four groups: cultivars Flame Prince, Cotender, Newhaven, Ruby Prince, Sun Prince, Jefferson, Camden and Jersey Queen were characterised as medium resistant to PPV; cultivars Loring, Blaze Prince, June Prince and Legend were classified as tolerant; cultivars Quachita Gold, O'Henry, Crest Haven, Biscos, Sentry, Fire Prince, Carogem, Carolina Belle, Redglobe and Harvester were rated as medium susceptible; and cultivars Bounty, Summer Prince, Gold Prince, Redhaven, Gala and Sunbrite were characterised as highly susceptible to PPV.

The incidence of Zucchini yellow mosaic virus (ZYMV) was monitored in the south Moravian region of the Czech Republic during 1997-2001. Crops of gherkin, squash, zucchini and cucumbers were found infected with ZYMV, manifested by symptoms of severe stunting and yellowing with deformed leaves and fruits. Twenty to eighty percent of infected plants were recorded. Six isolates of ZYMV from four localities were differentiated on indicator plants; three of them were maintained as being typical for differences in pathogenicity. Overwintered weed species were tested for the presence of the virus. ZYMV was detected by ELISA in one plant of Tripleurospermum maritimum out of 46 tested, and in two plants of Stellaria media out of 29 tested in 2001. Such tests were repeated in 2002, and ZYMV was detected in three plants of T. maritimum out of 45 tested, in three plants of S. media out of 52, and in two plants of Trifolium repens out of 17 tested. The virus was successfully transmitted from T. maritimum, S. media and T. repens to indicator plants of Cucurbita pepo convar. giromontiina. Back-transmission of ZYMV was proved by ELISA, electron microscopy and symptoms. T. maritimum was found to be a new natural host of ZYMV.

Plum pox virus (PPV) is widely distributed in plums and myrobalans in western, central and easternBohemia, in north-western, central and north-easternMoravia of theCzechRepublic. In southernBohemia and partly also in southernMoravia there is only a low and sporadic incidence. Naturally growing plums and myrobalans, and plums growing along roads were found to be the main sources and reservoirs of PPV infection. This high incidence in naturally growing plum and myrobalan trees makes it impossible to grow plum cultivars that are susceptible to PPV; only resistant cultivars can be grown in this country. In blackthorns the occurrence of PPV is limited to the regions with high and long-term presence of the virus. Therefore, we can conclude that blackthorn is not the primary, but a secondary source of PPV. On the other hand, sweet and sour cherries at localities of central and westernBohemia, and of southernMoravia are PPV-free. Till now the presence of strain PPV-C was not proved in theCzechRepublic. Strain PPV-M was proved only in two plum and one damson trees. It was also found in one apricot and one peach orchard planted with imported nursery material. Strain PPV-M appears to have been introduced recently and is absent from or has a very low incidence in spontaneous PPV hosts, while the widespread and long-term dissemination of strain PPV-D may indicate that it originated in the Czech Republic.

From 1997 to 2000 the distribution of Apple stem grooving virus (ASGV) in selected intensive apple orchards was investigated at six sites in the Czech Republic. Detection of ASGV was by ELISA. The virus was found to be commonly distributed; it was detected in over 50 % of the tested trees. Some cultivars were infected up to 100 %, while several were found to be virus-free. A high incidence of infected trees appeared in both older (20 years) and younger (7 to 8 years) orchards. This emphasizes the necessity to initiate the certification of the health condition of nursery material according to the system recommended by the EPPO.

In 1999, split vegetation tops in onions, cv. Stutgartska, grown in the surroundings of Prague, Bohemia, were observed. Such plants developed two or more bulbs from one originally planted. In addition, irregular and asymmetrical drying of the apical parts of outer skirts and in some cases scaling of the outer parts of divided bulbs occurred. Such divided bul bs suffered su bstantially higher damage during storage in winter; premature budding and grow the of individual parts was frequent. Further splitting of the vegetation tops into two to five parts and followed by premature budding was observed in individual parts of divided bulbs. Ultrathin sections were prepared from roots and leaves of divided bulbs. Phytoplasma bodies were found in tissues of both roots and leaves. Such bodies were not found in roots and leaves of healthy looking and undivided bulbs.

Myrobalan latent ringspot virus (MLRSV) was purified from the extract of infected Chenopodiumquinoa Willd., using n-octanol, differential centrifugation, and saccharose gradient centrifugation. Electron microscopy showed 28 nm large isometric particles in a sample of purified virus. Antiserum to MLRSV was prepared by immunisation of rabbits with intravenous injections of antigen in combination with intramuscular injections of antigen with Freund's adjuvant. The antibody titer of the obtained antiserum was determined by drop-precipitation method to be 1:1024. Immunoglobulins against MLRSV were isolated from antiserum with ammonium acetate, caprylic acid, and precipitation with ammonium sulphate. The isolated immunoglobulins (IgG) were conjugated with alkaline phosphatase. The optimal dilution of IgG for detection of MLRSV using ELISA was 1×10-3μg/ml which was also the optimal dilution of conjugated IgG. Using this dilution of antibodies, the absorbance of samples from MLRSV-infected plants of C. quinoa varied between 0.71 and 1.45, while absorbance of samples from healthy plants (control) was 0.01 to 0.07.

Commercialisation of Biotech/GM (Biotech) crops started in 1995. Not only field crops, but also horticultural transgenic crops are under development and are beginning to be commercialised. Genetic engineering has the potential to revolutionise fruit tree breeding. The development of transgenic fruit cultivars is in progress. Over the past 20 years an international public sector research team has collaborated in the development of HoneySweet plum which is highly resistant to Plum pox virus (PPV) the most devastating disease of plums and other stone fruits. HoneySweet was deregulated in the USA in 2010. HoneySweet (aka C5) has been evaluated for eleven years (2002-2012) in a regulated field trial in the CzechRepublic for the resistance to PPV, Prune dwarf virus (PDV), and Apple chlorotic leaf spot virus (ACLSV), all of them being serious diseases of plum. Even under the high and permanent infection pressure produced through grafting, PPV has only been detected in HoneySweet trees in several leaves and fruits situated close to the point of inoculum grafting. The lack of infection spread in HoneySweet demonstrates its high level of PPV resistance. Co-infections of PPV with PDV and/or ACLSV had practically no influence on the quantity and quality of HoneySweet fruit which are large, sweet, and of a high eating quality. In many respects, they are superior to the fruits of the well-known cultivar Stanley. Many fruit growers and fruit tree nurseries in the CzechRepublic are supportive of the deregulation of HoneySweet plum to help improve the plum production and control the spread of PPV.

Results of research aiming at monitoring of climate changes impact on plant pathogens distribution such as Zucchini yellow mosaic virus (ZYMV), quarantine Plum pox virus (PPV) and quarantine phytoplasma European stone fruit yellows (ESFY) are presented here. ZYMV has spread from Northern Italy across Austria up to Central Moravia and Bohemia. PPV has been continuously spreading from the lowlands of Central Bohemia and Moravia up to plains. Later, from the sixties and seventies of the last century, due to climate warming and human activities the virus quickly spread to uplands, foothills and mountains of the Czech Republic. Phytoplasma ESFY was spreading in a manner similar to ZYMV in the eighties of the twentieth century from Northern Italy and currently is affecting mainly apricot and peach trees in Southern Moravia.

A survey on the occurrence of plum pox virus (PPV) in plums, myrobalans, blackthorns, apricots and peaches was carried out in the South Moravian region along the Austrian border. Results of tests by ELISA and evaluation of PPV symptoms showed only scattered or isolated occurrence of PPV. This situation can be used for gradual elimination of PPV in the South Moravian region.