Superoxide and hydrogen peroxide levels in apricot trees infected by European stone fruit yellows phytoplasma

An epidemiological study on European stone fruit yellows (ESFY) phytoplasmas infecting Prunus fruit trees was carried out from 1994 to 2000 in Languedoc‐Roussillon (southern France). The spread of the disease was monitored for 7 years by visual observation of symptoms and by PCR detection of the phytoplasma in an experimental orchard planted with apricot hybrid seedlings. This indicated that aerial vectors were responsible for disease spread, and that transmission rates were low at the beginning of the spread. Seventy thousand homopteran insects were captured within and in the surroundings of highly ESFY‐infected apricot orchards, of which about 10 000 were used in PCR and nested‐PCR assays with universal ribosomal and ESFY‐specific nonribosomal primers to detect ESFY phytoplasmas. The other insects were confined in cages for trials of transmission to test plants. ESFY phytoplasmas could not be detected by PCR in any of the leafhopper species captured but could be detected in the psyllid Cacopsylla pruni caught on Prunus domestica and Prunus cerasifera rootstock suckers of apricot trees and on Prunus spinosa. Nested PCR revealed ESFY phytoplasmas in one individual of the deltocephalid Synophropsis lauri captured on an apricot tree. Transmission trials confirmed the role of Cacopsylla pruni as the ESFY phytoplasma vector in France. When apricot seedlings were used as bait plants from April to November during two consecutive years, no natural transmission could be demonstrated. However, one out of 50 apricot seedlings left for the whole year in the orchard became infected. An early spring ESFY infection is in agreement with both the natural transmission results and the life cycle of Cacopsylla pruni.

PCR-RFLP and sequence analysis of a non-ribosomal fragment for genetic characterization of European stone fruit yellows phytoplasmas infecting various Prunus species

Apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) are among the most economically important plant diseases that are caused by phytoplasmas. Phylogenetic analyses revealed that the 16S rDNA sequences of strains of each of these pathogens were identical or nearly identical. Differences between the three phytoplasmas ranged from 1.0 to 1.5% of nucleotide positions and were thus below the recommended threshold of 2.5% for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. However, supporting data for distinguishing the AP, PD and ESFY agents at the species level were obtained by examining other molecular markers, including the 16S-23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments. The three phytoplasmas also differed in serological comparisons and showed clear differences in vector transmission and host-range specificity. From these results, it can be concluded that the AP, PD and ESFY phytoplasmas are coherent but discrete taxa that can be distinguished at the putative species level, for which the names 'Candidatus Phytoplasma mali', 'Candidatus Phytoplasma pyri' and 'Candidatus Phytoplasma prunorum', respectively, are proposed. Strains AP15R, PD1R and ESFY-G1R were selected as reference strains. Examination of available data on the peach yellow leaf roll (PYLR) phytoplasma, which clusters with the AP, PD and ESFY agents, confirmed previous results showing that it is related most closely to the PD pathogen. The two phytoplasmas share 99.6% 16S rDNA sequence similarity. Significant differences were only observed in the sequence of a gene that encodes an immunodominant membrane protein. Until more information on this phytoplasma is available, it is proposed that the PYLR phytoplasma should be regarded as a subtype of 'Candidatus Phytoplasma pyri'.

Five new cultivars of French prune (Prune d'Ente) and 11 cultivars or new clones of greengage (Reine Claude) were bud‐inoculated with European stone fruit yellows (ESFY) phytoplasmas and evaluated for their host response in the nursery over a period of 6 years. The ESFY infection was confirmed by specific polymerase chain reaction (PCR) detection and 4′,6‐diamidino‐2‐phenylindole (DAPI) staining in all Prunus domestica cultivars tested which were, thus, all susceptible to ESFY phytoplasmas. The new Prune d'Ente cultivars were more sensitive as they generally developed ESFY symptoms and showed a mortality of 19%. The fruit weight was significantly reduced. No mortality was observed with Reine Claude genotypes and only two cultivars exhibited weak ESFY symptoms. A new sensitive method was developed to measure the impact of ESFY phytoplasmas on the shoot growth. A significant growth reduction was found in all Prune d'Ente cultivars but only in four out of 11 Reine Claude genotypes. Therefore, the latter were found to be tolerant of ESFY infection.

Between 1994 and 1998 a field study was conducted to identify plant hosts of the European stone fruit yellows (ESFY) phytoplasma in two apricot growing regions in southern and southwestern France where the incidence of apricot chlorotic leaf roll was high. A total of 431 samples from 51 different plant species were tested for the presence of phytoplasmas by PCR using universal and ESFY-specific primers. ESFY phytoplasma was detected in six different wild growing Prunus species exhibiting typical ESFY symptoms as well as in symptomless dog rose bushes (Rosa canina), ash trees (Fraxinus excelsior) and a declining hackberry (Celtis australis). The possible role of these plant species in the spread of ESFY phytoplasma is discussed. PCR-RFLP analysis of ribosomal DNA amplified with the universal primers was carried out to characterize the other phytoplasmas found. Thus, elm yellows phytoplasma, alder yellows phytoplasma and rubus stunt phytoplasma were detected in declining European field elm trees (Ulmus carpinifolia Gled), in declining European alder trees (Alnus glutinosa) and in proliferating Rubus spp. respectively. The presence of rubus stunt phytoplasma in great mallow (Malva sylvestris) and dog rose was demonstrated for the first time. Furthermore, the stolbur phytoplasma was detected in proliferating field bindweed (Convolvulus arvensis) and a previously undescribed phytoplasma type was detected in red dogwood (Cornus sanguinea). According to the 16S rDNA-RFLP pattern this new phytoplasma belongs to the stolbur phytoplasmas group.

Plant diseases caused by phytoplasmas have an increasing importance all over the world for fruit growers. Lately, phytoplasma diseases occur on many fruit species and are responsible for serious losses both in quality and quantity for fruit production. Apricot phytoplasma disease (Candidatus Phytoplasma prunorum) was reported first from France in Europe in 1924. Then, the pathogen spread in all European apricot-growing areas. In 1992, the disease was identified in Hungary first. Based on the growers’ signals, serious damage of Ca. Phytoplasma prunorum (Seemuller and Schneider, International Journal of Systematic and Evolutionary Microbiology, 54, 2004, 1217–1226; formerly: European stone fruit yellows (ESFY) phytoplasma) could be observed in several stone fruit orchards in the famous apricot-growing area nearby the town of Gonc, northern Hungary. Field examinations were started in 2009 in the infested stone fruit plantations in Borsod-Abauj-Zemplen County, mainly in Gonc region, which is one of the most important apricot-growing areas in Hungary, named “Gonc Apricot-growing area”. Our goals were to diagnose the occurrence of Ca. Phytoplasma prunorum on stone fruits (especially on apricot) in the North Hungarian growing areas by visual diagnostics and to confirm data by laboratory polymerase chain reaction (PCR)-based examinations. All the 40 collected samples were tested in laboratory trials and in 22 samples from apricot, peach, cherry, sour cherry and wild plum were confirmed the presence of phytoplasma (ESFY). Field investigations were done in a western Romanian apricot plantation, and the presence of apricot phytoplasma disease was confirmed. It was the first finding that Ca. Phytoplasma prunorum occurred in the western part of Romania. On the basis of these observations, it seems evident that the notable losses caused by Ca. Phytoplasma prunorum is a new plant health challenge for fruit growers to manage, especially for apricot producers in Hungary and other central European countries.

A Pilot Study Investigating the Relationship Between Diet and Oxidative Stress in Infants Receiving Therapy for Acute Leukemia

‘Candidatus Phytoplasma prunorum’ was detected for the first time in Tunisia in apricot trees that showed early leaf reddening in autumn, off-season growth in winter followed by dieback, and bore small and tasteless fruits. Phytoplasma was detected by nested PCR using the universal phytoplasma primer pairs R16mF2/R1 and R16F2n/R2. An amplification product of the expected size (1.2 kbp) was obtained from samples collected from symptomatic but not from symptomless apricot trees. Amplicons subjected to RFLP analysis after digestion with endonucleases MseI and RsaI, gave patterns similar to those reported for the European stone fruit yellows phytoplasma (ESFY, 16SrX-B). Identification was further confirmed by PCR using ESFY-specific primer pairs (ECA1/ECA2).

The extent of changes in sperm structure and function affect the success of fertilization ultimately during the pathway to ovum in the female reproductive tract. The success of AI with frozen-thawed semen varies in dairy and beef breeds of bovine because of differed alterations in sperm during transport in female tract after insemination. To our knowledge, no report is available comparing the changes in dairy and beef sperm leading to death in female tract. Therefore, this study was aimed to investigate the changes in motility, generation of reactive oxygen species (superoxide and hydrogen peroxide), and their relation to sperm death [asymmetry (apoptosis) and rupture of plasma membrane] of dairy and beef frozen-thawed semen during incubation at 37°C for 24 h. This incubation was aimed to mimic the environment of female reproductive tract. Frozen dairy semen (n = 4 bulls) was procured from a Canadian breeding station, whereas beef semen was collected from breeding beef bulls (n = 3; 5 replicates), diluted with Tris-based extender (composition was same as used in dairy semen), cooled to +4°C over 90 min, and cryopreserved by programmable freezer using standard rate as used in dairy semen. Two straws per replicate were thawed at 37°C from both types of semen, pooled separately, and incubated at 37°C for 24 h in capped tubes. Each pooled semen sample was evaluated for motility with CASA, superoxide (O2–, and hydrogen peroxide (H2O2) radical using HE/YoPRO and H2DCFDA/PI assay, respectively, and asymmetry of plasma membrane using YoPRO/PI assay through flow cytometric analysis at 0, 2, 4, 6, 12, and 24 h of incubation. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) was used to analyse the data as 2 × 6 factorial model for 2 types of semen (dairy and beef) and 6 time points using time as repeated measure. A threshold limit of 30% was considered for motility and live sperm to get optimum fertility. Sperm motility remained higher (P < 0.05) than threshold limit till 6 h in dairy (50.95 ± 2.62%) and 2 h in beef semen (30.28 ± 6.95%). Dairy semen possessed more (P < 0.05) nonapoptotic sperm without O2– (HE–/YoPRO–) till 6 h of incubation than beef semen. The increase in apoptotic sperm containing superoxide radical (HE+/YoPRO+) over time was more (P < 0.05) in beef semen till 6 h of incubation. The rise in dead sperm containing H2O2 (H2DCFDA+/PI+) was recorded more in beef than in dairy semen until 6 h of incubation. Live sperm without apoptosis (YoPRO–/PI–) were higher until 24 h in dairy (49.36 ± 4.56%) compared with beef semen (24.89 ± 3.85%), whereas viable sperm with apoptosis (YoPRO+/PI–) were found similar in both types of semen over time. In conclusion, dairy frozen-thawed semen possessed more live sperm without reactive oxygen species (superoxide and hydrogen peroxide) until 6 h of incubation than did beef semen. The decrease in superoxide radical was more in dairy than in beef semen. Dead and apoptotic sperm increased more in beef frozen-thawed semen over time during incubation. This inference suggests performing the insemination late near ovulation with beef frozen-thawed semen because of less viable life than dairy semen.

Disorder of physiological signaling functions of reactive oxygen species(ROS) superoxide and hydrogen peroxide and reactive nitrogen species (RNS) nitric oxide and peroxynitrite is an important feature of diabetes mellitus type 1 and type 2. It is now known that hyperglycemic conditions of cells are associated with the enhanced levels of ROS mainly generated by mitochondria and NADPH oxidase. It has been established that ROS stimulate many enzymatic cascades under normal physiological conditions, but hyperglycemia causes ROS overproduction and the deregulation of ROS signaling pathways initiating the development of diabetes mellitus. On the other hand the deregulation of RNS signaling leads basically to a decrease in NO formation with subsequent damaging disorders. In the present work we will consider the pathological changes of ROS and RNS signaling in enzyme/gene regulated processes catalyzed by protein kinases C and B (Akt/B), phosphatidylinositol 3′-kinase (PI3-kinase), extracellular signal-regulated kinase 1/2 (ERK1/2) and some others. Furthermore we will discuss a particularly important role of several ROS-regulated genes and adapter proteins such as the p66shc, FOXO3a and Sirt2. The effects of low and high ROS levels in diabetes will be also considered. Thus the regulation of damaging ROS levels in diabetes by antioxidants and free radical scavengers must be one of promising treatment of this disease, however,because of the inability of traditionalantioxidative vitamin E and C to interact with superoxide and hydrogen peroxide,new free radical scavengers such as flavonoids, quinones and synthetic mimetics of superoxide dismutase (SOD) should be intensively studied.

Epithelial-mesenchymal transition (EMT) represses cell adhesion associated genes, making cells more migratory. Snail is the master gene that regulates the EMT process by down regulating tight junction proteins, such as E-cadherin, and up-regulating mesenchymal genes, such as vimentin. Cell migration can also be regulated by the Rho family of GTPases such as Rac1, which can induce reactive oxygen species (ROS), specifically superoxide, via the NADPH oxidase (Nox) pathways. ROS, specifically hydrogen peroxide has been shown to mediate EMT induced by MMP-3. The objective of this study is to determine whether Snail Transcription factor contributes to tumor progression via ROS and Rac1 and to study the effect(s) of antioxidants on Snail signaling. We utilized androgen-dependent LNCaP and androgen-independent 22Rv1 and ARCaP cell lines overexpressing Snail to examine Rac1 activity in vitro. ROS (superoxide and hydrogen peroxide) levels were examined in vitro and in vivo. Western blot analysis of EMT markers, cell migration on collagen and Real Time PCR Array to assay oxidative stress enzymes was also performed. We found that LNCaP, 22Rv1 and ARCaP cells overexpressing Snail displayed increased Rac1 activity while LNCaP and ARCaP cells with Snail overexpression expressed higher levels of superoxide and hydrogen peroxide in vitro. Furthermore, ARCaP cells overexpressing Snail injected into nude mice showed higher tumorigenicity as compared to ARCaP Neo control cells and excised tumors displayed increased levels of superoxide and hydrogen peroxide ex vivo. Real Time PCR Array revealed significant increase in key enzymes involved in oxidative stress such as aldehyde oxidase I and peroxidasin. Treatment with the hydrogen peroxide scavenger, N-acetyl cysteine (NAC), could partially revert Snail-mediated EMT in ARCaP cells as evidenced by re-expression of E-cadherin after 3 days. The muscadine grape skin extract (MSKE) antioxidant decreased ROS levels and Rac1 activity and reverted Snail-mediated EMT as seen by decreased vimentin, re-induction of E-cadherin, and decreased cell migration. This is the first report of Snail regulation of ROS by regulating oxidative stress enzymes, leading to EMT and tumor progression. We are also showing novel data that Snail can regulate cell motility via Rac1 activation. Therefore, targeting Snail-mediated EMT with antioxidants offers a promising therapy to reduce prostate cancer progression in future. Research supported by NIH Grants: 1P20MD002285 and G12RR03062. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5332. doi:1538-7445.AM2012-5332

This study focused on quantification of 'Candidatus Phytoplasma prunorum' in apricot trees exhibiting uneven symptoms of European stone fruit yellows. For the research, 17 trees showing early leaf bud break in one part of the crown were selected in winter season, and 13 trees showing leafroll in older part of the shoots and no symptoms in younger part of the same shoots were selected in summer season. Absolute quantification of phytoplasmas was performed by real-time PCR in samples collected from asymptomatic and symptomatic parts. The results showed, that phytoplasma could not be always detected in asymptomatic parts, where 2 out of 17 samples from asymptomatic parts from winter season were negative while in summer season 6 out of 13 samples from asymptomatic parts were negative. Comparison of phytoplasma quantities between symptomatic and asymptomatic plant parts then showed significantly higher quantities in symptomatic parts from samples collected in summer season and no quantity difference in samples collected in winter season.

Hydrogen peroxide (H2O2) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants: asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that ‘Candidatus Phytoplasma prunorum’, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant. H2O2 was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H2O2 was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases. Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H2O2 and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.

‘Candidatus Phytoplasma prunorum’, the causal agent of European stone fruit yellows (ESFY), is one of the most important pathogens causing considerable economic losses in stone fruit orchards. This study evaluated trees infected and noninfected by phytoplasma ESFY of 16 apricot varieties grown in an orchard in Lednice (Czech Republic) between the years 2008-2014. Pomological traits, phenophases, pollen germination and seed viability were analysed as well as the presence of ‘Ca. P. prunorum’ in pollen, flower organs, fruit flesh, immature seeds and seedlings by nested PCR. One of the most detrimental impacts was the decreased fruit set of infected trees which occurred in 12 out of 16 studied varieties reaching an average fruit set decrease of 16.1%. Pollen germination rates also suffered, showing an average decrease by 11.78% in infected trees. In the analysis of some traits, overall significant differences were observed between the infected and noninfected trees. However, for all analysed traits in different varieties, both positive and negative influences of phytoplasma ESFY were observed. The presence of ‘Ca. P. prunorum’ in infected trees was confirmed in flower parts (only in peduncle in 70.2% of cases) and fruit parts (immature fruit flesh in 42.0% and milky kernels in 26.2% of cases), however, neither in seedlings nor in pollen.

In the last twenty years, three phytoplasma diseases were identified in Hungary, viz. European Stone Fruit Yellows (ESFY) (caused by Candidatus Phytoplasma prunorum), pear decline (caused by Candidatus Phytoplasma pyri), and apple proliferation (caused by Candidatus Phytoplasma mali). Candidatus Phytoplasma prunorum was isolated from apricot, peach, plum and japanese plum. Cacopsylla pruni the vector of ESFY was also isolated and identified. Infection of Candidatus Phytoplasma pyri was diagnosed from pear and Candidatus Phytoplasma mali was found on apple and pear. The three phytoplasmas cause different damages on their host plants. The most economically important phytoplasma disease is the ESFY. It seriously impairs apricot and japanase plum trees. After infection of apricots and japanese plums show yellowing and defoliation, and within a few years die in apoplexy-like symptoms. The disease on japanese plum is so severe that this fruit practically can not be cultivated in Hungary. Pear decline is the most serious problem especially in intensive pear plantations. The vector Cacopsylla pyri, C. pyrisuga and C. pyricola can be found in almost all pear orchards. Because of the regular presence of psyllids in intensive pear orchards the insecticide control is necessary. Apple proliferation is not an important disease in Hungary. All of our isolations of ’Candidatus Phytoplasma mali’ occured in organic orchards and record was not available in Hungary lately.