Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status a cellular adaptive response occurs requiring functional chaperones, antioxidant production and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring plasma reduced and oxidized glutathione, as well as pentosidine, protein carbonyls lipid oxidation products 4-hydroxy-2-nonenal and F2-isoprostanes in plasma, and lymphocytes, whereas the lymphocyte levels of the heat shock proteins (HSP) HO-1, Hsp72, Sirtuin-1, Sirtuin-2 and thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. Plasma GSH/GSSG showed a significant decrease in type 2 diabetes as compared to control group, associated with increased pentosidine, F2-isoprostanes, carbonyls and HNE levels. In addition, lymphocyte levels of HO-1, Hsp70, Trx and TrxR-1 (P < 0.05 and P < 0.01) in diabetic patients were higher than in normal subjects, while sirtuin-1 and sirtuin-2 protein was significantly decreased (p < 0.05). In conclusion, patients affected by type 2 diabetes are under condition of systemic oxidative stress and, although the relevance of downregulation in sirtuin signal has to be fully understood, however induction of HSPs and thioredoxin protein system represent a maintained response in counteracting systemic pro-oxidant status. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

Author(s): Raspagliesi, D.; Licciardello, G.; Lombardo, A.; Rizza, S.; Bar-Joseph, M.; Catara, A.

Over 4,000 Clementine trees grafted on trifoliate orange, spaced at 2 x 3 m, were inoculated 1 yr after planting with four different isolates combinations of Citrus exocortis viroid (CEVd), Citrus viroid III (CVd-III) and Hop stunt viroid (HSVd). As expected, 12 yr after inoculation, all the trees containing CEVd showed bark cracking and/or scaling, whereas those with only CVd-III were healthy in appearance. The trees inoculated with CVd-III alone had the largest average circumference of both scion and rootstock, fruit quality reached high standards and yields were more than twice that of a conventional orchard. Those inoculated with CVd-III+HSVd, with CEVd+CVd-III, and CVd-III+HSVd+CEVd were smaller. The phenotypic response of host plant was not related with the titer of CVd-III. Citrus trees are often naturally infected with various combinations of viroids inducing different phenotypic reactions (4). Some variants of the Citrus viroid III have been studied for many years as potentially useful, due to their capability of reducing the canopy of citrus grafted on trifoliate orange without any detrimental effects. In fact. There are reports of enhanced yield per canopy volume (3). Therefore, the name of Citrus dwarfing viroid (CDVd) has been proposed to replace CVd-III, since it is more descriptive of the host phenotype induced by the infection (5). High density citrus plots, obtained through the inoculation of citrus viroids in different combinations have been established in Italy for several years with variable results, depending on the scionrootstock combination, the viroids and the pedoclimatic conditions (1). In order to obtain some clarity, a large orchard of over 4,000 Comune Clementine grafted on trifoliate orange was established in the south of Italy close to Lamezia (Calabria region, Catanzaro province), GPS coordinates N 38°08' 33,9''/ E 016°09' 01,6''. In spring 1992, about 4,500 seedlings of trifoliate orange were grafted in a commercial nursery with buds of certified Clementine SRA, and after 2 yr they were planted at a spacing of 3 x 2 m (1,666 trees /ha). The trees were inoculated 1 yr after planting by grafting onto the each scion two pieces of bark taken from source viroid-infected plants. There were four different isolate combinations of Citrus exocortis viroid (CEVd), Citrus viroid III (CVd-III) and Hop stunt viroid (HSVd). The plants were maintained standard agricultural practices and periodically checked for symptoms, fruit quality and growth. Normal fertilizer and pesticide applications for the area were performed, and irrigation was applied by a sprinkler irrigation system. From the fourth year, the trees were pruned regularly in order to allow good lighting and fruiting and to reduce pest control sprays. In the first 5 yr, pruning tools were disinfected with sodium hypochlorite solution between trees to prevent cross transmission of viroids. During the summer 2005, 40 randomly selected trees of Comune Clementine, showing different phenotypes, were monitored for symptoms, growth, yield and viroid content by RT-PCR. CVd-

A new approach for the rapid discrimination of mild and severe Citrus tristeza virus (CTV) isolates was developed by a sequential process made up of the DAS-ELISA immunological test followed by fluorescence-based Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP) analysis.The new method helps both in locating CTV infected trees and in preliminarily typing of the virus isolates, thus saving both time and resources.Partial p18 gene amplification products of CTV-RNAs directly extracted from infected leaves or recovered from ELISA plates are presented.Specific profiles of forward and reverse strands were obtained when biologically distinct CTV isolates were analysed both directly and following ELISA.The CE-SSCP method is simple, highly sensitive and highly reproducible, and can process a large number of samples for a variety of needs.Applied in a combined process, ELISA and CE-SSCP helped simultaneously to detect the virus and to collect useful information on the genetic diversity and structure of the CTV population present in a territory.

Viroids are small circular RNA molecules, 246 to 400 nt long, which infect several crop plants and can cause diseases of economic importance.Citrus spp.are the hosts in which the highest number of viroids has been recovered.Citrus exocortis viroid (CEVd), causal agent of citrus exocortis disease, is responsible for several losses in citrus crops.Little is known about the molecular and cellular mechanisms by which viroids infect plants and produce symptoms.To deepen this knowledge, changes in the gene-expression profile during the early (pre-symptomatic) and the last (post-symptomatic) stage of Etrog citron infection by Citrus exocortis viroid (CEVd) were investigated using a citrus cDNA microarray.MaSigPro analysis was performed, and based on their expression profile along time, genes were divided into five clusters.These results will allow us to have a clearer idea about the changes in the plant transcriptome that are associated with symptom expression and/or with specific plant defense mechanisms.

Over 4,000 Clementine trees grafted on trifoliate orange, spaced at 2 x 3 m, were inoculated 1 yr after planting with four different isolates combinations of Citrus exocortis viroid (CEVd), Citrus viroid III (CVd-III) and Hop stunt viroid (HSVd).As expected, 12 yr after inoculation, all the trees containing CEVd showed bark cracking and/or scaling, whereas those with only CVd-III were healthy in appearance.The trees inoculated with CVd-III alone had the largest average circumference of both scion and rootstock, fruit quality reached high standards and yields were more than twice that of a conventional orchard.Those inoculated with CVd-III+HSVd, with CEVd+CVd-III, and CVd-III+HSVd+CEVd were smaller.The phenotypic response of host plant was not related with the titer of CVd-III.

To study the host plant-viroid interaction, green bark samples of Troyer citrange, sour orange and Alemow seedlings inoculated with a Citrus viroid-IIIb (CVd-IIIb) source were processed by a rapid and sensitive RT-SYBR Green I-based real time assay to assess molecular events taking place at an early stage of infection.The messenger RNA (mRNA) differential display technique was applied for the identification and isolation of genes whose transcription was altered significantly in Etrog citron leaves infected by CVd-IIIb.Seven of them, known to be related to plant stress defense, were selected to evaluate the variation of the expression level in Troyer citrange, sour orange, and alemow seedlings by Northern Blot analysis and reverse-transcriptase quantitative real time PCR (RT-qPCR).Both methods were valuable, but the latter was more sensitive.The seven genes were detected, in the three rootststocks, as up-or down-regulated.The inoculation of CVd-IIIb in Troyer citrange seedlings modified the expression of metallothionein (MT), alcohol dehydrogenase (ADH), ethylene-responsive binding protein (EREBP), regulator of gene silencing (RGS), peroxidase (PRX) and CONSTANS-like at a lower level than in Etrog citron but with a similar pattern.Alemow seedlings showed a slight over-expression of MT and ADH; in contrast, PRX was severely down regulated.Sour orange seedlings did not reveal any signal with the exception of PRX and CONSTANS, which were up regulated as in Etrog citron.RT-qPCR allowed the detection of the genes, which escaped the Northern Blot analysis, showing in sour orange the same pattern (over-expression) as Etrog citron for ADH, EREBP and AP; whereas MT and RGS were down regulated.In alemow seedlings, EREBP, RGS, AP and CLP showed a reduction of the expression level as well as AP in Troyer citrange.

In recent years, Citrus tristeza virus (CTV) has often been reported in Italy.Interestingly enough, despite the wide use of sour orange rootstock the typical inverse pitting commonly associated with CTV infection has only been reported on trees derived from introduced budsticks of citrus varieties, and recently on some 30 yr-old Sanguinello sweet orange trees.Recently, thousands trees of different varieties at distant locations were examined for inverse pitting without success.Preliminary studies showed that the isolate of CTV infecting two representative Sanguinello orange trees was different from other CTV isolates found close to this area which do not induce inverse pitting.All the sources indexed on Mexican lime seedlings showed typical vein clearing after only 10 days, whereas only sour orange, sweet orange and grapefruit inoculated with the Sanguinello sources showed stunting, yellowing, small and/or cupped leaves and vein corking to different degrees.The gene coding for p23 was amplified and the nucleotide sequences were determined in both directions.BLAST analysis showed a nucleotide identity of 99% with seedling yellow strains like BaraoB, Val-CB and C271-2.Results confirm that more than a single introduction of CTV could have occurred in Italy over the years.

A rapid and sensitive real time reverse transcription-PCR (RT-PCR) assay based on SYBR Green I chemistry was developed for the quantitative detection of Citrus viroid III (CVd-III) in citrus samples. CVd-III titre was determined at different times in green bark of sour orange, Troyer citrange, trifoliate orange and alemow seedlings inoculated with a CVd-IIIb source. Ten weeks after inoculation the viroid was detected in the four species, without substantial differences in viroid titre among them. Nine weeks later an overall increase of viroid titre was observed. The copy number of CVd-III in sour orange and Troyer citrange was monitored up to 52 weeks after inoculation and a further increase of viroid titre was observed at 35 weeks. For validation purposes, field samples were tested from 58 citrus trees with mixed infections of CVd-III, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), as well as from healthy controls. Based on the sensitivity (100%), specificity (96.7%), accuracy (99.2%) and repeatability (Cohen's kappa index 0.98) of the assay, it is suggested that its employment in breeding programmes would be helpful in the evaluation of host resistance and viroid accumulation in plants.

Citrus cultivation in China has increased since the late 1970s, with China now having the largest area of citrus in culture in the world that is spread in 22 provinces and municipalities. Hunan Province has undergone a program to become one of the major citrus producers in China. Poncirus trifoliata is the main rootstock, so citrus viroids are a limiting factor for further citriculture development. In mainland China, only the presence of Citrus exocortis viroid (CEVd) has been reported from Etrog citron indexing, sPAGE (sequential polyacrylamide gel electrophoresis) analysis (2), and reverse transcription (RT)-PCR (3). Three viroid-like RNAs, a1, b1, and d, based on sPAGE patterns were detected years ago in our laboratory in budsticks received from Sichuan Province. To identify different viroids and determine their distribution, a survey has been undertaken. Field trees showing stunting, bark scaling and cracking of the rootstock, and poor yield were tested using biological indexing and PCR for the most frequent citrus viroids. Samples from six trees of a local sweet orange variety and three of a Clementine variety introduced from abroad, both grafted on P. trifoliata and showing a variable degree of bark scaling and cracking, were collected near Changsha and in the County of Xin Ning at the end of summer 2006. Small pieces of bark were inserted in stems of young E. citron budwood grafted on rough lemon and maintained in a warm greenhouse (24 to 32°C). Indexing on E. citron showed mild epinasty and leaf roll typical of citrus viroid infections. To identify specific viroids, bark was ground to a fine powder with liquid nitrogen and total RNA was extracted with TRIZOL Reagent (Invitrogen, San Diego, CA) and tested by RT-PCR to detect CEVd, Hop Stunt viroid (HSVd), and Citrus viroid III (CVd-III), as well as to identify the cachexia variants of HSVd. Four primer pairs were used to test the RNA extracts by RT-PCR (1). All samples were infected by HSVd, eight with CVd-III, and six with CEVd. The cachexia variants of HSVd were detected in four of nine samples. Mixed infections were as follows: one sample had CEVd and HSVd, eight had HSVd and CVd-III, and five were infected by the three viroids. A second sampling 3 months after inoculation gave the same amplification patterns. The results show that at least three viroids are present in citrus orchards in Hunan Province. To our knowledge, this is the first report of cachexia variants of HSVd and CVd-III in China. The common occurrence of these viroids supports the need for proper indexing of mother trees and a specific shoot tip grafting program to create healthy budwood sources to provide healthy plants. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes, 20:105, 2006. (2) L. Han et al. Viroids. CSIRO Publishing, Melbourne, 283, 2003. (3). Q. Hu et al. Acta Bot. Sin. 39:613, 1997.

The use of viroids as tools to obtain tree dwarfing for high density citrus planting needs careful risk assessment, and is a process which should include evaluating dwarfing sources for the potential to generate viroid genome mutations and for the induction of new diseases.In order to study the stability of Citrus viroid IIIb (CVd-IIIb) populations in natural field conditions we have analyzed in two different host species the progeny of CVd-IIIb derived from the same original source.Viroid RNA populations from seven trifoliate orange and one Troyer citrange seedling, bark-inoculated in 1983 with the same Italian CVd-IIIb isolate were analyzed.Fulllength double-stranded CVd-IIIb cDNAs were synthesized by RT-PCR using primer pairs designed from the upper strand of the central conserved region or the terminal right region.Analysis of about ten full-length cDNA clones obtained from each trifoliate orange tree showed similar RNA populations comprised of a predominant species of 294 nt which was identical to the CVd-IIIb variant previously reported.Similar results were obtained with the CVd-IIIb population derived from Troyer citrange.These results demonstrate the high level of sequence stability of CVd-IIIb in two different hosts grown for a long period in the field.

Citrus exocortis viroid (CEVd) and Citrus viroid-IIb (CVd-IIb) are the etiological agents of citrus exocortis and citrus cachexia diseases, which cause economically important damage in citrus crops. For this reason they are both included in citrus certification programs. The need to speed up the detection of pathogens has stimulated progressive improvement in and application of appropriate technology. In the present study we have devised real-time RT-PCR protocols using the intercalating dye SYBR-Green I for fast detection of these two pathogens in field samples infected with a mixture of viroids. Primer pairs designed from highly conserved regions of the genome of different variants of each viroid amplified DNA fragments of 83-bp (CEVd) and 133-bp (CVd-II), which were detected by the increasing fluorescence observed during the reaction. The analysis of the melting temperatures (Tm) allowed for the discrimination of the amplified products generated from the two distinct viroid templates. The presence of products specific to CEVd, cachexia and non-cachexia CVd-II was always confirmed by visualization with polyacrylamide gel electrophoresis. The evidence indicates that real-time RT-PCR appears to be a useful tool for fast and reliable diagnosis of citrus viroids. Index words : Viroids, citrus, real-time RT-PCR, SYBR-Green, melting temperature. Viroids are transmitted mainly by vegetative propagation and the principal way of control is the use of viroid-free propagation material. For this purpose the availability of simple and rapid methods for their detection is critical. Currently, conventional techniques used for citrus viroid diagnosis include biological tests with indicator plants (Etrog citron and Parson’s Special mandarin), sequential polyacrylamide gel eletrophoresis (sPAGE) (7) and reverse-transcription polymerase chain reaction (RT-PCR) (3, 8), but the need for decreasing the time needed for detection of these pathogens in field samples is still a primary focus of research in this branch of plant pathology. Recently real-time RT-PCR has been applied to the detection of Potato spindle tuber viroid (PSTVd) (1). In this paper we report the use of two protocols based on the use of a method employing SYBR-Green I real-time RT-PCR for the diagnosis of pathogenic citrus viroids. Real-time PCR allows, in contrast to conventional assays, the monitoring of de novo generation of PCR products by detecting the increase in fluorescence as the amplification progresses. As SYBR-Green I detects all dsDNA, including primer dimers and other undesired products, specific amplicons can be distinguished by their melting curve, which is dependent on GC content, length, and sequence (6). For the current study, five citrus plants (trifoliate orange, Etrog citron, Orlando tangelo, Fino Iniasel and Kamarina lemons) infected with a mixture of different viroids and one healthy plant of Arizona 861-S1 Etrog citron were used as source material to establish the protocols. The viroid isolates were previously characterized by bioassay on Etrog citron and Parson’s Special mandarin and by sPAGE analysis and found to contain CEVd, cachexia or non-cachexia CVd-II, as well as other viroids. In order to define optimal conditions, total nucleic acids were phenol-extracted from 10 g of green bark and partially purified by CF11 cellulose column chromatography Sixteenth IOCV Conference, 2005—Short Communications 457 (2), concentrated by ethanol precipitation and finally re-suspended in 100 μl sterile distilled H 2 O. Primers were designed using the software FAST PCR (v.3.7 Inst. of Biotechnology, Univ. of Helsinki, Helsinki, Finland) (5, 9). Criteria chosen to design high efficiency primers using this program included amplification from the central conserved region of each viroid and similar annealing temperatures for primers, in order to perform both protocols contemporaneously in the same reaction. The primer pair designed for CEVd (Genbank Accession No: M34917) consisted of the 20-mer cev-a (5’GGGAAACCTGGAGGAAGTCG-3’), homologous to nucleotides 98-117 and the 18-mer cev-b (5’-tgtttctccgctggacgc-3’), complementary to nucleotides 159-180. The primers for CVd-II (Genbank Accession No: AF213484, AF213503) were the 18mer hsv-a (5’-GGCAACTCTTCTCAGAATCC-3’), homologous to nucleotides 84-101 and the 19-mer hsv-b (5’-GTCTCACTCGAAGAGCCAG3’), complementary to nucleotides 199-216. The effectiveness of the primer pairs was checked previously by RT-PCR (8). The cDNA preparation for each viroid was synthesized using 1 μl of nucleic acid extracts in a 24 μl RT reaction containing RT buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 10 mM DTT), 0.4 μM of complementary primers cev-b or hsv-b, 0.5 mM each of dATP, dTTP, dCTP and dGTP, 25 U of Rnasin (Promega) and 20 U of M-MLV reverse transcriptase (Promega). Reverse transcription reactions were conducted at 42°C for 30 min. The threshold cycle (Ct), i.e. the cycle in which the level of fluorescence of the sample increases above the background, was chosen as one of the criteria to determine the presence of the desired target. Melting curve analysis was used as a second criterion to determine target presence or absence, since each PCR product exhibits a characteristic peak (Tm) at its maximum melting temperature. SYBR Green I-based PCR analyses were performed using the iCycler iQ Multicolor Real-time Detection System (BIO-RAD). Real time reactions were performed in a final volume of 25 μl containing 2 μl of cDNA and 1 μl of each primer at 0.4 μM final concentration and the iQ SYBR Green Supermix 2X (BIORAD Laboratories). Real-time PCR used the following amplification parameters for both CEVd and CVd-II: 5 min of HotStar Taq polymerase activation at 95°C, followed by 40 cycles of PCR at 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s. Melting curve analysis was conducted under the following conditions: 1 min denaturation at 95°C, 1 min annealing at 60°C, followed by 180 cycles with annealing temperatures increasing in 0.2°C increments (10 sec each) beginning at 60°C with continuous reading of fluorescence. Real-time PCR products were also analyzed by Fig. 1 Amplification plots generated in the real-time RT-PCR reactions with the primer pair cev-a/cev-b (left) and hsv-a/hsv-b (right). 458 Sixteenth IOCV Conference, 2005—Short Communications 6% PAGE to confirm the identity of the targets and show the absence of nonspecific PCR products. CEVd as well as cachexia and non-cachexia CVd-II variants were detected by the Real-Time RT-PCR using SYBR green I intercalating dye and the indicated primer pairs. Ct values between 12.9 and 23.9 and between 14.5 and 24.9 were observed for CEVd and CVd-II, respectively (Fig. 1). Melting curve analysis performed at the end of amplification revealed Tm peaks of 87.26°C ± 0.10 for CEVd and 85.26 ± 0.09 for CVdII respectively (Fig. 2). Healthy and no template controls did not show increased fluorescence before the 36 th or 37 th cycle, and melting curve analysis for these samples revealed a peak at about 75°C (Figs. 1, 2). Analysis by 6% PAGE showed amplicons of the expected size, 83 bp for CEVd and 133 bp for CVd-II without detectable non-specific products. No amplification products were observed from healthy and water controls (Fig. 3). As expected we obtained positive results for all the infected samples tested, with a useful increase of fluorescence and consequently low Ct values. In contrast, healthy controls gave a very delayed Ct, a common problem associated with the use of SYBR-Green which may be due to the formation of primer dimers (4), thus providing a clear distinction of positive from negative samples. The synthesis of amplicons from both cachexia and non-cachexia CVd-II isolates indicates that we can amplify different variants of CVd-II but we cannot distinguish pathogenic from non-pathogenic forms due to a low annealing temperature used in the reaction. Designing primers from sequence in the variFig. 2. Melting curves obtained for CEVd (left) and CVd-II (right). Fig. 3. Polyacrylamide gel electrophoresis (5%) analysis of real-time RT-PCR products of CEVd (on the left) and CVd-II (on the right) compared with a DNA molecular size marker (50bp DNA Step Ladder, Promega). Isolates were: 1. trifoliate orange; 2. Etrog citron; 3. Fino iniasel lemon; 4. Kamarina lemon; 5. Orlando tangelo; 6. healthy control; 7. RT H20; 8. H20. Sixteenth IOCV Conference, 2005—Short Communications 459 able domain (which contains five mutations which distinguish the two groups of variants) could resolve the two groups, but also could give erratic results in RT-PCR. The primer pairs used in this protocol in conjunction with a specific probe designed from sequence in the variable domain could allow differentiation of those mutations, coupling high efficiency of amplification with increased specificity. In conclusion, these results show that real-time RT-PCR can be a useful tool for fast and reliable detection of CEVd and CVd-II, providing the possibility to run both reactions simultaneously using the same annealing temperature. The method is rapid and easy to perform since, in routine analyses, it does not require electrophoresis, which is time-consuming and uses the mutagenic agent ethidium bromide.