Citrus Exocortis Research Articles

DETECTION OF POSPIVIROIDS IN THE CZECH REPUBLIC AND THEIR DISCRIMINATION BY RESTRICTION ANALYSIS

Potato spindle tuber viroid (PSTVd), a member of the genus Pospiviroid, is a quarantine pest in the European Union and its presence in ornamental solanaceous crops represents a serious threat to susceptible vegetable and potato crops. Since the year 2007 the Czech Plant Protection Service (PPS) carries out a survey for PSTVd on ornamental solanaceous species. Within the first year of the survey, several new hosts of PSTVd were identified. Also, a new species of Pospiviroid was detected in the Czech Republic. At the beginning of the survey, a method for detection of PSTVd recommended in the EPPO diagnostic protocol was adopted. Reverse transcription-polymerase chain reaction with a semi-specific primer pair was carried out in the diagnostic laboratory of PPS while the sequencing of PCR products was performed in an external laboratory. Due to this time consuming process, which proved inefficient for the needs of rapid application of phytosanitary measures, the laboratory developed a faster procedure. The entire genomic sequences of all viroids which contained primer identical segments were downloaded from the GenBank database and aligned to search for viroid-specific restriction sites. A combination of four enzymes was chosen to distinguish the detected viroids. Restriction analysis was successfully tested and applied for the PSTVd identification. Later on, after confirmation of other pospiviroids detected for the first time in the Czech Republic (Tomato apical stunt viroid and Chrysanthemum stunt viroid on Solanum jasminoides and Citrus exocortis viroid on Solanum jasminoides and Verbena A— hybrid), the diagnostic method was improved to detect a wide range of Pospiviroid species with the introduction of primers detecting the five most common pospiviroids in the ornamental solanaceous plants in Europe. Similarly, for identification of the amplified PCR products, a new set of restriction enzymes was identified.

PERFORMANCE OF THE TUNISIAN 'MALTAISE DEMI SANGUINE' SWEET ORANGE INOCULATED WITH CITRUS EXOCORTIS VIROID (CEVD) AND CACHEXIA VIROID (CVIIB) ON EIGHT ROOTSTOCKS

The performance of ‘Maltaise demi-sanguine’ sweet orange infected with Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd,Variant CVIIb) was evaluated on eight different rootstocks (sour orange, ‘Carrizo’ citrange, volkamer lemon, ‘Cleopatra’ mandarin, ‘Swingle’ citrumelo, ‘Rangpur’ lime, alemow and trifoliate orange) at the INRAT station in El Gobba, Cap Bon region, Tunisia. The trees were planted in 2005 and fruit production and fruit quality and size were evaluated every year since 2007. For non-inoculated trees, the trunk circumference was larger for plants grown on citrumelo, volkamer lemon and ‘Carrizo’ citrange than for those grown on sour orange. Trees on volkamer lemon also had a larger canopy volume. Infection with HSVd-CVIIb caused severe stunting and low production on trees grafted on alemow, and to a lesser extent reduced trunk diameter and production of trees on ‘Swingle’ citrumelo, whereas inoculation with CEVd or HSVd had little effect on the growth and yield of trees propagated on the other rootstocks. The only effect of HSV-CVIIb infection on fruit quality consisted of increased juice weight in plants propagated on ‘Rangpur’ lime and acidity in plants on volkamer lemon and decreased total soluble solids in plants on sour orange.

Citrus Viroids: Symptom Expression and Performance of Washington Navel Sweet Orange Trees Grafted on Carrizo Citrange.

Citrus are natural hosts of several viroid species. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of two well-known diseases of citrus, exocortis and cachexia. Other viroids have been found to induce specific symptoms and different degrees of stunting in trees grafted on trifoliate orange and trifoliate orange hybrids. A field assay was initiated in 1989 to establish the effect of CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) on Washington navel sweet orange trees grafted on Carrizo citrange rootstock. Here we report the effect of viroid infection on symptom expression, tree size, fruit production and quality evaluated from 2004 to 2007. Vegetative growth was affected by viroid infection with height and canopy volume being reduced. No bark scaling symptoms were observed in CEVd-infected trees albeit they presented lesions and blisters in the roots. Bark cracking symptoms were consistently observed in CBCVd-infected trees that were smaller with enhanced productivity and fruit size. No major effects were found as a result of infection with CBLVd, HSVd, or CDVd. The quality of the fruits was not affected by viroid infection, except for the low diameter of the fruits harvested from HSVd-infected trees. An interesting effect was identified in terms of tree productivity increase (yield/canopy volume) as a result of infection with CEVd, CDVd, and especially CBCVd.

A new disease inCitrus aurantifoliain Oman, “sudden decline”, is associated with a pathogen complex including a 16SrII group phytoplasma

Witches’ broom disease of lime is a devastating disease in the Sultanate of Oman. In recent years a new disease has been observed, which promotes a sudden decline in Citrus aurantifolia (acid lime). The etiological agent of this new disease has not yet been determined. In this study the presence of phytoplasmas and RNAs was evaluated in plants showing the sudden decline symptomatology. The results reveal the presence of a complex of at least three different pathogens: 16SrII phytoplasmas, Citrus tristeza virus and Citrus exocortis viroid.

First Report of Tomato apical stunt viroid in Tomato in Italy.

During a survey conducted in October 2013 in tomato greenhouses in Diano Marina (Imola Province, northwest Italy), in a single greenhouse, unusual disease symptoms were observed in four out 1,400 (~0.3%) of tomato plants cv. Ingrid, grafted on 'Beaufort' rootstock. Symptoms including shortened apical internodes associated with tiny, deformed, and brittle chlorotic leaves, while ripe fruits appeared reduced in size and pale red. Samples of leaves from the four plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Tomato spotted wilt virus, Cucumber mosaic virus, Alfalfa mosaic virus, Tomato/Tobacco mosaic viruses, and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (potygroup test). None of the tested viruses were detected in the four leaf samples. In addition, PCR tests for begomoviruses and phytoplasmas were also negative. In a host range study, the original symptoms, consisting mainly of stunting and chlorosis, were reproduced within ~10 days in tomato seedlings (Momor line), mechanically inoculated at two true leaves stage with sap extract obtained from the four symptomatic tomato plants, whereas no symptoms were observed in Chenopodium amaranticolor, C. quinoa, Nicotiana tabacum (cv. Xanthi nc), N. glutinosa, or Phaseolus vulgaris (cv. Borlotto rosso). Total RNAs extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from symptomatic samples were tested in RT-PCR using pospiviroids generic primers PSTVd-32/33 (1), designed to amplify the whole genome of Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd), and Columnea latent viroid (CLVd), and CEVd-FW/RE primers, designed to amplify the whole genome of Citrus exocortis viroid (CEVd) and TASVd (3). Each of the four samples yielded amplicons of the same size (364 bp) with both primer combinations. The identity of the viroid was then determined by sequencing, on both strands, amplicons obtained from the four symptomatic plants at MWG (Ebersberg, Germany). Sequences obtained were identical, showing the highest nucleotide identity (99.7%) with the TASVd isolate Sj1 (AM777161), identified in Germany on Solanum jasminoides. The sequence was deposited in GenBank (Accession No. HG916812) and the field isolate named To1-IT. Two other cases of pospiviroid infection in tomato in Italy have been reported so far and the viroid species detected were PSTVd (2) and CLVd (4), respectively. To our knowledge, this is the first report of TASVd infecting tomato in Italy. The origin of this infection is still unclear, although based on the biological properties and sequence similarity, the To1-IT isolate probably originated from an ornamental species, most likely S. jasminoides, as recently reported for other tomato TASVd isolates, according to their biological and genetic features (5).

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.

Citrus exocortis viroid. [Distribution map

Abstract A new distribution map is provided for Citrus exocortis viroid. Pospiviroidae: Pospiviroid. Hosts: Citrus spp. and trifoliate orange ( Poncirus trifoliata ). Information is given on the geographical distribution in Europe (Austria, Belgium, Cyprus, Czech Republic, France, Germany, Greece, Italy, Sardinia, Sicily, Montenegro, Netherlands, Portugal, Russia, Slovenia and Spain), Asia (China, Chongqing, Fujian, Henan, Hubei, Hunan, Liaoning, Shandong, Sichuan, Zhejiang, India, New Delhi, Maharashtra, Indian Punjab, Indonesia, Iran, Iraq, Israel, Japan, Honshu, Jordan, Korea Republic, Lebanon, Malaysia, Oman, Pakistan, Philippines, Saudi Arabia, Syria, Taiwan, Thailand, Turkey, United Arab Emirates, Vietnam and Yemen), Africa (Algeria, Cameroon, Cote d'Ivoire, Egypt, Ethiopia, Ghana, Libya, Madagascar, Mauritius, Morocco, Mozambique, Nigeria, Reunion, Sierra Leone, Somalia, South Africa, Sudan and Tunisia), North America (Canada, New Brunswick, Mexico, USA, Arizona, California, Florida, Louisiana and Texas), Central America and Caribbean (Cuba, Guadeloupe, Jamaica and Trinidad and Tobago), South America (Argentina, Bolivia, Brazil, Sao Paulo, Chile, Colombia, Peru, Suriname, Uruguay and Venezuela) and Oceania (Australia, New South Wales, Northern Territory, Queensland, Victoria, Cook Islands, Fiji, French Polynesia, New Zealand, Papua New Guinea and Samoa).

Affordable and reliable plant sap-mediated template preparation for the detection of various phytopathogens by PCR assay

Plant pathogens like ‘Ca. Liberibacter’, phytoplasma, viruses and viroids cause diseases to almost all economically important plants. A simple and fast sap-mediated polymerase chain reaction (PCR) method for the detection of these pathogens infecting various plant hosts is described in the present study. Sap from selected plants was drawn aseptically on parafilm, from the mid-rib of young leaves. Depending on the type of host plant, sap was diluted to optimal concentration before PCR analysis. ‘Ca. Liberibacter’, Citrus tristeza virus (CTV) and Citrus exocortis viroid (CEVd) infecting citrus, and ‘Ca. Phytoplasma’ infecting pepper and sandal trees were tested by sap-mediated PCR. The reliability of this procedure was evaluated by comparing the findings with previously described protocols. The sap-mediated nucleic acid template preparation for PCR assay is devoid of laborious nucleic acid extraction and expensive chemicals. Hence the present method is rapid, economical and so can be employed for diagnosis of large number of plant samples.

Simultaneous detection of CTV, CEVd and HSVd using Arizona 861 S1 Citron and RT-PCR

R. Camps, M. Castro, and X. Besoain. 2014. Simultaneous detection of CTV, CEVd and HSVd using Arizona 861 S1 Citron and RT-PCR. Cien. Inv. Agr. 41(2): 255-261. Citrus tristeza virus (CTV), Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are pathogens that affect citrus trees in Chile. The simultaneous use of a rapid diagnostic method with the effective cleaning of infected budwood is a powerful preventative method for controlling viral diseases. The objective of this study was the multiple detection of CTV, CEVd, and HSVd, through bio-amplification in Arizona 861-S1 citron plants, combined with two incubation temperatures and multiple RT-PCR in two steps. Citron indicator plants were inoculated with the three pathogens, both individually and simultaneously, with four un-inoculated plants as controls. After four months of incubation at the two different temperatures, both the multiple and individually inoculated plants showed symptoms associated with these pathogens, which were subsequently corroborated through the use of multiple RT-PCR in two steps. This diagnostic method could be employed as an efficient, cost-effective alternative to the methods that are currently in use for detecting CTV, CEVd and HSVd, nevertheless its effectiveness should also be evaluated with tests using different isolates of these pathogens.

OCCURRENCE, PREVALENCE AND DISTRIBUTION OF CITRUS VIROIDS IN URUGUAY

The occurrence of Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd) and Citrus viroid VI (CVd-VI) in Citrus spp. cultivated in six provinces of Uruguay was surveyed in 2008- 2009 and 2009-2010 growing seasons using Northern blot hybridization. Sixty two per cent of surveyed trees were infected with either single or mixed viroid inocula, the latter being more abundant. CBCVd and CVd-VI were not detected in the samples analyzed. HSVd showed the highest prevalence (92%) among infected plants followed by CDVd (50%), CEVd (23%) and CBLVd (19%). The most frequently observed viroid combination was HSVd+CDVd. Our results showed that CEVd, CBLVd, HSVd, CDVd are widespread throughout citrus orchards in Uruguay and highlight the presence of mixed viroid infections.

Complex mixtures of viroids identified in the two main citrus growing areas of Iran

The citrus industry in Iran is based on graft propagation of local and imported cultivars on a limited number of rootstocks such as sour orange, Mexican lime and Bakraee, a local natural hybrid. Infection of the available cultivars with graft-transmissible agents, viroids in particular, may have deleterious effects on the productivity of citrus and, more importantly, limits the choice of rootstocks. Rootstocks other than sour orange and Mexican lime will be needed as a consequence of the introduction and spread of Citrus tristeza virus (CTV) in the country. In the present study, two cv. Sanguinello sweet orange and two cv. Duncan grapefruit trees were selected on the basis of the bark scaling symptoms observed on the trifoliate orange and Troyer citrange rootstocks. Analysis by sPAGE and Northern blot hybridization of nucleic acid preparations of Etrog citrons graft-inoculated with these sources revealed the presence of several viroids: Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the causal agents of exocortis and cachexia, respectively, as well as Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd) and Citrus bark cracking viroid (CBCVd). These viroids were sequenced and compared with the corresponding reference sequences. To extend these results, samples from different citrus cultivars were collected in the two main citrus growing regions and analyzed for viroids. Of the 49 samples tested, 22 were viroid-positive, witnessing the occurrence of cultivars infected with CEVd, HSVd, CBLVd, CDVd, or CBCVd as mixed infections in the surveyed regions. One sample was also co-infected with the newly described Citrus viroid V (CVd-V).

Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

First Report of Grapevine yellow speckle viroid-1 and Hop stunt viroid Infecting Grapevines (Vitis vinifera) in India.

During March through July 2012, 10 to 15% of the Vitis vinifera cultivars Thompson Seedless and Anab-e-Shahi exhibited yellow leaf spots and flecks, shortened internodes, and tiny yellow leaves in vineyards of the Bijapur, Doddaballapur, and Kolar districts of Karnataka State, India. These are the major grapevine cultivation regions in India. Samples were collected from four different plants from each district (12 samples in total) and RNA was extracted using 2X CTAB buffer (1). Presence of Grapevine yellow speckle viroid1 (GYSVd-1, genus Apscaviroid) was tested by reverse transcription (RT)-PCR with primer pair PBCVd100C/194H (4) for the amplification of a 220-bp region of the genome. In agarose gel electrophoresis, five samples showed amplicons of the expected size. These amplicons were cloned and sequenced. BLAST analysis confirmed the presence of GYSVd-1. Based on this data, the full-length genome of GYSVd-1 was amplified by RT-PCR using primer pair 341M (5'-CACTCGCGGGGCGCGTTGGA-3') and 342P (5'-CAATCCCCGGAACCCCCGCT-3') and the amplicons were cloned and sequenced. Sequence analysis revealed two sequence variants namely Kar-1 (GenBank Accession No. AB742222) and Kar-2 (AB742223) with 98% and 99% identity to GYSVd-1 variants IXc (X87913) and II (X87906), respectively. GYSVd-1 variants Kar-1 and Kar-2 clustered in two distinct phylogenetic sub-clades. All 12 samples also tested positive for Hop stunt viroid (HpSVd, genus Hostuviroid) in two separate sets of RT-PCR using HSV-78P (5'-AACCCGGGGCAACTCTTCTC-3') and HSV-83M (5'-AACCCGGGGCTCCTTTCTCA-3'); and HSV-7P (5'-AATTCTCGAGTTGCCGC-3') and HSV-220M (5'-CGAACCGAGAGGTGATGCCA-3'), with the expected size of 303 and 213 bp, respectively (3). Sequence analysis of the amplicons confirmed the presence of HpSVd. Alignment of HpSVd nucleotide sequences obtained from the 12 samples showed the presence of a single type of sequence variant, namely Ind-2 (AB742225). BLAST analysis showed 99% sequence identity of Ind-2 with a HpSVd variant isolated from a 100-year-old grapevine in China. All 12 grapevine samples were also tested for the presence of Australian grapevine viroid (AGVd, genus Apscaviroid), Grapevine yellow speckle viroid 2 (GYSVd 2, genus Apscaviroid), and Citrus exocortis viroid (CEVd, genus Pospiviroid) by RT-PCR as described previously (2). None of the samples showed any positives. Northern blot assay using appropriate digoxigenin-labeled riboprobes performed as described previously (2) further confirmed RT-PCR results. Positive controls for RT-PCR and Northern blot were obtained from viroid-infected grapevines maintained in the greenhouse. GYSVd-1 and HpSVd were detected in symptomatic and symptomless plants. Hence, the symptoms observed in the vineyard cannot be attributed to viroid infection. More work is needed to identify the causal agent(s) of the decline of Thompson Seedless and Anab-e-Shadi cultivars.

Variants of Hop stunt viroid Associated with Mulberry Vein Clearing in Iran

AbstractSamples of mulberry leaves showing vein clearing, yellow speckle and leaf deformation symptoms were collected in the Fars Province of Iran. Grafting of buds from affected trees onto a number of healthy mulberry rootstocks resulted in transmission of the pathogen 40 days postinoculation. Likewise, mechanical inoculation of sap from affected leaves resulted in induction of similar symptoms in mulberry seedlings. Total nucleic acid was extracted from leaf tissues and subjected to reverse transcription polymerase chain reaction (RT‐PCR) using Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd)‐specific primers, followed by sequencing of PCR products. RT‐PCR with HSVd primers amplified a 302‐ or 305‐bp product from affected samples, but none from healthy plants. CEVd was not detected in affected trees. HSVd was also found in graft‐ and mechanically inoculated plants. Sequence analyses showed three variants of HSVd in symptomatic mulberries related to plum and peach variants of this viroid with 94.9% identity, but with only 93% identity to Lebanese and Italian mulberry isolates. This is the first report of HSVd associated with vein clearing in mulberry in Iran.

Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens

A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for ‘Candidatus Liberibacter asiaticus’ (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected both RNA and DNA pathogens in the same dilution series as singleplex assays and yielded similar quantitation cycle values. Taken together, high throughput extraction and multiplex RT-qPCR assays reported in this study provided a rapid and standardized method for routine and simultaneous diagnosis of different RNA and DNA citrus pathogens.

Identification and characterization of known and novel viroid variants in the Greek national citrus germplasm collection: threats to the industry

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection.

A Leafhopper-Transmissible DNA Virus with Novel Evolutionary Lineage in the Family Geminiviridae Implicated in Grapevine Redleaf Disease by Next-Generation Sequencing

A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.

Identification and characterization of viroids in 'Navelina ISA 315' sweet orange

To date, seven species of viroids have been described infecting Citrus. However, only Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus dwarfing viroid (CDVd) have been reported in this crop in Brazil. The aim of our work was to identify and characterize viroids infecting plants of 'Navelina ISA 315' sweet orange cultivar grafted on 'Rangpur' lime from field experiments located in Bebedouro, SP, that showed gumming in the bark and wood symptoms. Biological indexing was done on the indicator host 'Clemelin 1120' tangor plants that reacted with typical gumming symptoms. Viroid infection was assessed by graft-inoculation of buds from the field trees on 'Etrog' citron plants grafted on 'Rangpur' lime, followed by RNA extraction and sPAGE analysis. RNAs were subjected to RT-PCR with primers for citrus viroids. The full-length amplified products were sequenced and compared to those available in the GenBank. The trees were found to be infected with cachexia (Ca) variants of HSVd-Ca or HSVd-Ca plus CDVd. The results indicate that efforts have to be made to increase and stimulate the indexing programs, to maintain plants healthy and to develop sanitary programs focused on reducing the spread of viroids and other graft-transmissible agents.

A noncoding plant pathogen provokes both transcriptional and posttranscriptional alterations in tomato

Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a β-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens.

In vitroshoot tip grafting in citrus species- A review

A retrospective overview of work done on the in vitro shoot tip grafting in citrus in the world was taken up. In vitro shoot tip grafting is the most reliable method to recover free citrus saplings from infected parental source. The technique of in vitro shoot tip grafting is a suitable method for the elimination of grafttransmissible diseasesin citrus. Preliminary studies showed that certain pathogens are difficult to be eliminate from mother plant like citrus exocortis and stubborn, which might be eliminated by a process of shoot tip grafting in-vitro. Plant obtained by micro-grafting do not have the same problems as nucellar plants such as reversion to the juvenile state, excessive thorniness, vigorous and upright habit of growth, slowness to fruiting, alternate bearing in early years and physical differences in fruit characteristics.