Citrus tristeza virus (CTV) is an aphid-borne pathogen that causes devastating disease. Due to its devastating effects reaching endemic, epidemic and pandemic levels , it was named as “tristeza” which means “sadness” in Spanish and Portuguese. CTV belongs to Closterovirus group with largest genome size of 19.3kb.CTV variants causing symptoms in citrus like stem pitting, quick decline and seed yellows, initially restricted to regions of Asia, Australia, South Africa, and South America, have been successively reported in other citrus areas, including California, Florida, and the Mediterranean region. The virus CTV is phloem limited and is reported to be transmitted by viruliferous aphids Toxoptera citricida, Aphis gossypii, A. citricola, T. aurantia. Detection and identification of CTV can be achieved using biological, serological, or molecular amplification tests. Biological indexing or indicator plants can be used for the extraction, diagnosis of viruses, and further detection by RT-PCR and ELISA. Trifoliate orange and its derivative cultivars can be used as a resistant rootstock against Quick Decline. Preimmunization with mild strain through cross protection is the best method of managing CTV.

Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host–virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.

My PhD thesis work of Citrus tristeza virus (CTV) purification was aimed to develop a rapid serological assay to replace biological indexing. The task turned difficult and was achieved after a lengthy struggle, rewarded by allowing (1) the rapid diagnosis of the first incidences of natural spread of a severe CTV-VT strain in our region and (2) finding that the CTV particle isolation protocol, with some modifications, was also useful for Beet yellows virus (BYV) particles, leading to their assignment in the Closterovirus group, the first group of elongated plant viruses with different modal lengths. Later, following the introduction of ELISA for large-scale diagnosis of tristeza-infected citrus trees, the CTV infection rates through the coastal citrus production areas were continually increasing, with many ELISA-positive samples appearing symptomless, prompting the need to develop strain-specific assays. Using CTV-VT cDNA fragments, as hybridization probes, the genetic diversity among local CTV isolates was demonstrated. With the emergence of the PCR technology, we developed a CTV-dsRNA cloning method based on the ligation of known oligonucleotide molecules to dsRNA ends and the use of complementary oligonucleotides for cDNA synthesis and PCR amplification. The method allowed the cloning of a cDNA molecule complementary to a defective dsRNA of 2.4kb with intact 5 and 3 ends of the CTV-VT genome. A list of publications, resulting from continuous collaborative work with local and foreign associates and students on the development and adaptation of novel CTV methodologies, is present.

The occurrence of viruses and viroids in ornamental citrus mother plants in Tuscany (Central Italy)

Antiserum prepared to a local isolate of grapevine virus A (GVA) was used in immunosorbent electron microscopy (ISEM) with decoration and in an unlabelled antibody enzyme-linked immunosorbent assay (ELISA) for the screening of grapevine sources for the presence of virus. GV A occurred extensively in grapevine plantings showing visual symp· toms of leafroll or indexing positive for leafroll. GV A was found not to be associated with either stem pitting or fleck symptoms. It occurred mostly in association with undecorated closterovirus (CV)-like particles. Limited ISEM with decoration of the CV-like particles using antiserum to a Swiss isolate of CV 2 200 nm, (CV type I) confirmed the presence of a second CV in local grapevine sources. In addition, it indicated the presence of a third CV -like particle longer than GVA. GVA together with undecorated CV-like particles were also detected in initially GVA-free LN-33 grapevines growing under field conditions and naturally infected with Ieafroll. GV A and CV type I were also present in the vine mealybug Planococcus ficus, following exposure to a grapevine source carrying these viruses. H'lwever, due to lack of CV type I antiserum, only GV A was confirmed in grapevine following controlled transmissions with P. ficus although undecorated CV -like particles were present. The reliability of the ELISA detection procedure appears to be influenced by seasonal fluctuations in GV A concentrations.

Viruses developed a strategy to counter-defence the posttranscriptional gene silencing mechanism (PTGS) based on the activity of silencing suppressor proteins. Citrus tristeza virus (CTV), a member of the genus Closterovirus , has two suppressor proteins (p20 and p23) that target the local RNA silencing response of the host. In GFP transient co-expression assays performed on Nicotiana benthamiana 16C plants, local suppressor activity of p23 and p20 was similar. Co-expression of both proteins from a mild or a stem pitting CTV isolate showed stronger local suppression activity than either suppressor alone, with an increased GFP transcript level six- (for Gp M) to nine-fold (for Gp 3a) higher than non-inoculated 16C plants, in parallel with low accumulation of siRNAs. Further, GFP brightness of leaves infiltrated with Agrobacterium cultures at an OD 600 of 0.5 was comparable to those infiltrated with OD 600 0.25. These findings indicate that combined action of p20 and p23 proteins results in enhanced suppressor activity.

Comparison of infection of Citrus tristeza closterovirus in Kinnow mandarin (Citrus reticulata) and Mosambi sweet orange (Citrus sinensis) in Pakistan

Leafroll is one of the most harmful viral diseases affecting grapevine worldwide. Historically, a dozen of viruses, named grapevine leafroll-associated viruses (GLRaVs), belonging to genera Closterovirus and Ampelovirus (Family Closteroviridae), have been found associated with the disease. Recent studies showed that GLRaV-4, -5, -6, -Pr, Deand -Car are in fact strains of the same virus species prompting taxonomic and nomenclatural revision of these viruses. Among these viruses, GLRaV-4 strain 5 has been reported from many viticultural areas in the world. In 2012-2013, surveys for virus detection disclosed the identification of GLRaV-4 strain 5 in 768 samples collected in Tuscany. The virus presence was ascertained by real-time (RT)-PCR.
\nGLRaV-4 strain 5 was found in 2 samples (0.63% rate, 2012) and in 4 samples (1.16%, 2013) in cvs Sangiovese and Canaiolo. Further molecular analysis revealed that one infected vine hosted a single GLRaV-5 infection, while other infected vines were infected with multiple viruses, including GLRaV-3, GVA, GFkV and GRSPaV.
\nThe GLRaV-4 strain 5 sequences obtained from the positive samples shared 96% nucleotide identities with the corresponding fragment of a reference GLRaV-5 isolate (GenBank accession No. JX559639.1). The nucleotide sequence was deposited in GenBank as accession No. KM252726. To the best of our knowledge this is the first report of the occurrence of GLRaV-4 strain 5 in Italian vineyards.

The brown citrus aphid, Toxoptera citricida (Kirkaldy), is a serious pest of citrus because it efficiently transmits citrus tristeza closterovirus (CTV). The invasion of Jamaica's citrus by T. citricida resulted in serious economic losses and the importation and consideration was given to the establishment of Lipolexis oregmae Gahan (Hymenoptera: Aphidiidae) in a classical biological control program. Prior to introducing L. oregmae, we conducted a survey to determine the abundance of Lysiphlebus testaceipes Cresson (Hymenoptera: Aphidiidae) in T. citricida. A high-fidelity PCR analysis was conducted using Lysiphlebus- and Lipolexis-specific ITS2 rRNA primers on alcohol-preserved brown citrus aphids and the results were positive for both parasitoids. The fortuitous establishment of L. oregmae in Jamaica was surprising, and was confirmed by rearing adults from parasitized aphids and obtaining taxonomic confirmation based on morphology. In addition, its identity was confirmed molecularly by cloning and sequencing 16S, 12S, and COI sequences from the Florida colony of L. oregmae and from Jamaican specimens. The three DNA sequences were 100% identical for specimens from both sources. The phylogenetic analysis using 16S sequences indicated a close relationship of the Florida and Jamaica populations of L. oregmae with L. gracilis. During 2005, an island-wide survey of citrus in six parishes confirmed that both L. testaceipes and L. oregmae commonly parasitize T. citricida, but that L. oregmae was the more widely distributed and abundant parasitoid. The pathway by which L. oregmae might have entered Jamaica is discussed.

In a survey of carnations grown in the Kangra valley of Himachal Pradesh, India, 17 out of the 35 varieties inspected showed mottling with grey streaks or red necrotic flecks. These symptoms were similar to those previously reported for Carnation necrotic fleck virus (CNFV) (Bar-Joseph et al. , 1979). Sap from leaves with symptoms was inoculated on to Dianthus caryophyllus , D. chinensis and D. barbatus. Symptoms produced on these Dianthus spp. indicator plants included mottling, vein clearing, flecking, grey streaks or red necrotic flecks. No symptoms were observed when Chenopodium spp. were inoculated. Using a polyclonal DAS-ELISA kit (Agdia Inc., Elkhart, IN, USA), CNFV was detected in Dianthus indicator plants and the following carnation varieties: Arthur Sim, Cabarett, Charmour, Dessio, Dusty Pink, Espana, Flair, Irma, Josh, LasPama, New Espana, Orange Triumph, Prinidello, Red Carso, Scania, Shocking Pink and White Candy. The virus was transmitted in a semipersistent manner by aphid species Myzus persicae , Aphis craccivora and Aphis gossypii from D. caryophyllus to D. barbatus , with infection confirmed by DAS-ELISA. When examined by transmission electron microscopy, leaf dip preparations showed flexuous virus particles 1400–1500 nm long. Reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted using degenerate primers specific to the closterovirus group (Karasev et al. , 1994) and an amplicon of the expected size ( ∼ 1000 bp) was generated. All these tests identified the virus as CNFV. This virus has been reported from many countries, including the USA, France, Italy and Yugoslavia, but this is the first report of CNFV infecting carnations in India.

Citrus tristeza Closterovirus (CTV) is widespread in major citrus-growing regions of the world often causing destructive diseases. Citrus samples were taken from orchards in the Croatian coastal region. CTV was detected in two symptomless field trees of Satsuma mandarins and one diseased lemon tree. Double-stranded RNA was isolated from the field trees and the dsRNA patterns were compared in polyacrylamide gels. The same dsRNA extracts were used as templates in RT-PCR experiments amplifying the CTV coat protein sequence. Amplicons were subjected to SSCP and RFLP analyses. The results indicate greater similarity between CTV isolates from Satsuma mandarins than between these two and the lemon isolate.

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

An extensive survey was carried out to assess, by ELISA, the occurrence of grapevine closteroviruses in several grape-growing areas of Greece, with particular regard to GLRaV-7. Samples were collected in commercial vineyards and varietal collections from apparently symptomless and symptomatic leafroll vines. GLRaV-7 was widely distributed and frequently associated with GLRaV-1 and GLRaV-3. ELISA was suitable for GLRaV-7 detection but the reagents utilized in this study must be improved.

Field surveys were carried out in the main sugar beet growing areas of Lebanon to assess the occurrence and distribution of viral diseases. A total of 1002 samples from 115 commercial fields were serologically assessed for Beet Necrotic Yellow Vein Furovirus (BNYVV), Beet Yellows Closterovirus (BYV), Beet Mosaic Potyvirus (BtMV), Beet Western Yellow Luteovirus (BWYV) and Cucumber Mosaic Cucumovirus (CMV). ELISA tests revealed that 39.5% of samples were infected with one or more viruses. BtMV was the most common (56.5%), followed by BYV (29.5%), BNYVV (17%) and BWYV (11%). Mixed infections were detected in 5.2% of the samples. CMV was not encountered. Direct tissue printing of plants infected with BtMV, BYV and BWYV on a nitrocellulose membrane gave very strong positive reactions.

Citrus Tristeza Closterovirus (CTV) induces mild and/or severe symptoms on Citrus species. It may cause death of trees if the rootstock-scion combination is susceptible. It has been found in other plant/virus combinations that transformation with partial or complete viral genes (e.g., coat protein genes) can confer resistance to the resulting transgenic plants. We previously reported A. tumefaciens mediated transformation and production of two sour orange (C. aurantium L.) plants expressing the coat protein gene of CTV, which was the first report of production of transgenic Citrus using a viral gene. However, in order to properly evaluate resistance, it is necessary to obtain as many transgenic Citrus plants from single transformation events as possible. Therefore, we are currently transforming grapefruit (Citrus paradisi) `Marsh' and `Star Ruby' and sweet orange (C. sinensis) `Valencia' with CTV coat protein genes. These species are susceptible to CTV and more amenable to transformation than sour orange. Epicotyl segments of etiolated seedlings were inoculated with A. tumefaciens strain EHA101 harboring binary plasmid pGA482GG containing the coat protein gene of mild Florida CTV strain T30 (CP-T30) or severe Florida strain T36 (CP-T36). Putatively transformed shoots were regenerated on selection medium containing kanamycin. Regenerated shoots were evaluated with GUS assays; those shoots positively identified by GUS were then evaluated with PCR. We have currently identified 17 `Marsh' grapefruit, 20 `Star Ruby' grapefruit, and seven sweet orange putatively transformed plants.

Ultrastructural observations were carried out on cells of Nicotiana benthamiana infected with a new mechanically transmissible isolate of Grapevine leafroll-associated virus 2 (GLRaV-2-4H), Vitis rupestris infected with the same isolate, V. vinifera cv. ‘Semillon’ infected with GLRaV-2 isolate Se, and an undetermined V. vinifera cultivar infected with Grapevine leafroll-associated virus 7 (GLRaV-7). GLRaV-2 and GLRaV-7 are members of the genus Closterovirus. Regardless of the host, both viruses appeared to multiply only in the phloem, affecting the cytology of differentiating sieve tubes, parenchyma and companion cells. Both GLRaV- 2 isolates induced the same type of ultrastructural modifications in the herbaceous host and Vitis, consisting primarily of membrane proliferation, formation of inclusion bodies and virus particle aggregates. Inclusion bodies were made up of clusters of membranous vesicles with a fibrillar content, surrounded by a single membrane, intermixed with loose aggregates of virus particles. The vesicles did not seem to derive from mitochondria in both GLRaV-2 and GLRaV-7 infections, thus setting a difference between these viruses and two other grapevine closteroviruses (GLRaV-1 and GLRaV- 3) previously studied. Virus particles of GLRaV-2 and GLRaV-7 were plentiful and accumulated in the cytoplasm and nuclei of both Vitis species investigated.

A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza closterovirus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0.47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA.

Kinetics of Accumulation of Citrus Tristeza Virus RNAs

Summary.Citrus tristeza closterovirus (CTV) isolates of several geographical origins were compared for variations in their coat protein (CP) gene by analysis of single‐strand conformation polymorphism (SSCP). The CP gene of 17 isolates was reverse transcribed, amplified by polymerase chain reaction (PCR), and 22 clones were inserted into a plasmid vector. These clones were sequenced and found to have between 91.7% and 99.8% sequence homology. Clones were amplified and the PCR products denatured and compared by SSCP analysis in 8% polyacrylamide gels. Using two different electrophoretic conditions, the patterns were different for 16 or 17 clones. Four pairs of clones (T36/T66, P1/Q2, 03/8Q, and E1/E2) differing by 10, 2, 1 and 1 nucleotides, respectively, could not be distinguished using either condition. When these clones were compared by SSCP after digestion with Eco91I (BstEII) three of the pairs (T36/T66, P1/Q2, and 03/8Q) could be differentiated, whereas the clones E1 and E2 (differing by 1 nucleotide) remained indistinguishable. Thus, SSCP analysis combining two electrophoretic conditions and restriction of eight clones with Eco91I allowed discrimination between 21 of the 22 CP gene clones selected.SSCP analysis may provide a procedure to identify and differentiate CTV isolates based on comparisons of several genes or gene regions. It is rapid and cheap and may drastically reduce the amount of sequencing necessary for accurate comparisons.

The complete, 19,226 nt sequence of the RNA genome from VT, a seedling yellows strain of citrus tristeza virus (CTV), was determined and found to have a genome organization identical with that of the previously determined CTV-T36 isolate, except that ORF 1 of CTV-VT was 70 nt shorter due to two widely separated 18 nt deletions. Sequence comparison of CTV-VT and CTV-T36 revealed approximately 89% identity throughout the ten 3' ORFs, but only 60-70% identity throughout ORF 1. The 5' nontranslated regions were only 60% identical whereas the 3' nontranslated regions were 97% identical. The transition between regions of similarity and deviation was gradual, suggesting that the sequence similarities and differences compared to CTV-T36 were unlikely to have arisen from a recent recombination event between a close T36 relative and a distantly related CTV isolate. This is the first attempt to compare in detail the variation between the genomes of two strains of a member of the closterovirus group. The observed deviation between the large RNA genomes of the two CTV strains is greater than that among different viruses of most other groups, raising the question of how to define the taxonomy of these viruses.