Are the viroid-like molecules the evolutionary link between the RNA and DNA world? We hope that the current and the future generations of citrus scientists will carry on the 80 years old journey of viroid research and that they will provide exciting answers, new discoveries, and even more questions for scientific advancement.Citrus exocorThis viroid , is a member of the genus Pospiviroid within the Pospiviroidae family. CEVd is the causal agent of exocortis disease characterised by bark scaling and dwarfing symptoms in sensitive citrus hosts commonly used as rootstocks and limiting crop production of the grafted commercial species. Since symptom expression takes 3-5 years to show on inoculated trifoliate orange Raf the biological characterization of CEVd usually relies on the use of indicator plants. Etrog citron has been widely used for indexing purposes, bio-amplification of the viroid RNA and strain characterization. Following infection with CEVd, Etrog citron displays a characteristic syndrome that includes severe stunting, leaf epinasty and midvein necrosis in 3-6 months. Furthermore, the effect of a severe isolate of CEVd on the gene expression of Etrog citron has been examined, using a citrus cDNA microarray approach that revealed that infection triggered important changes in chloroplast, cell wall, peroxidase and symporter activities. However the changes in gene expression found in CEVd infected Etrog citron may not be necessarily responsible for the bark scaling symptoms that are the most characteristic exocortis syndrome in trifoliate orange. Four trifoliate orange seedlings that were graft inoculated with a severe strain of CEVd and were planted in a field plot in June 1993,led grow lights developed the characteristic bark scaling symptoms, yellowing of the twigs and remained stunted as compared with the non-inoculated controls.
In 2010 plant material was collected in order to accomplish a gene expression analysis using the same genome-wide 20-K cDNA microarray developed under the Citrus Functional Genomic Project . For that, the gene expression profiles of the CEVd infected trifoliate oranges and uninfected control ones were compared. Subsequently, Significance Analysis of Microarrays was performed to identify those genes differentially expressed in both conditions and lastly, they were classified with gene ontology analysis by using the Blast2GO tool . As expected, results showed similarities and differences with those obtained for Etrog citron that could account for some of the differences found in the symptomatology. We developed a semi-automated, high throughput, RNA extraction and purification procedure optimized for citrus tissues. The system utilizes the SPEX SamplePrep’s Cryo-station and Geno Grinder 2010, and the Applied Biosystems’ MagMAXTM Express-96 along with a modified 5x MME-96 Viral RNA Isolation Kit. The detection of viral RNA with reverse transcription quantitative real time polymerase chain reaction requires high quality RNA as defined by concentration, purity, and integrity. RNA concentration and purity were assessed by spectrophotometry at 260 nm, and 260/280 and 260/230 ratios, respectively. The RNA concentration of 304 citrus samples ranged from 14.4 to 886.0 ng/μl with an average of 180.082 . The majority of the samples had concentrations ≥50ng/μl while 89.1% of the samples had concentrations of 50-400 ng/μl. The RNA purity ratios were higher than the desirable 1.8 for all samples tested with a mean value of 2.404 while 98% of the samples had 260/280 ratio ≥2.0. The 260/230 ratios had a broader variation in comparison to the 260/280. The 260/230 ratio mean value was 1.883 . The majority of the samples had the desirable 260/230 ratio of 1.8-2.0 .
The remaining 27% of the samples had 260/230 ratio of 1.0-1.7 and the remaining 11% had 260/230 ratio ˂1.0. Subsequent experimentation with adjusted grinding and washing buffers significantly improved and standardized the 260/230 ratios to the desirable values of 1.8-2.0.The RNA integrity of 23 samples was evaluated by 118 RT-qPCR reactions targeting the mRNA of the NADH dehydrogenase citrus gene. The mean Ct value of the RT-qPCR was 21.948 with maximum and minimum values 28.5 and 16.29, respectively. The cost of supplies for the presented RNA extraction and purification procedure was estimated at US$4.03 per sample. Citrus vein enation disease is characterized by woody galls on the trunk and vein enations on the leaves of susceptible citrus species. This disease is graft transmissible and naturally spread by aphid species in a persistent manner. With the aim of identifying the putative virus associated with vein enation, small interfering RNAs from three different field citrus trees that tested positive to citrus vein enation by indexing and that are located in Valencia, Tenerife and Gran Canaria, Spain, were analyzed by next generation sequencing Illumina technology. Bioinformatic analysis of individual samples allowed the identification of a new Luteoviridae present in the three samples. Bioinformatic analysis using the CLC Genomic Workbench, Velvet, Geneious and Bowtie software allowed the reconstruction of a sequence of 5077 nucleotides, corresponding to a new viral species for which the name Citrus vein enation virus is proposed. Open reading frames were identified coding for an hypothetical protein, the polymerase, the aphid transmission protein and the coat protein. Protein homology analyses for these ORFs showed similarities with Luteoviridae members: 44% , 66% , 62% and 70% , respectively. Specific primers and a TaqMan probe based on the new sequence were designed for real-time RTPCR detection of the agent.
The method allowed the successful detection of this virus in plant material and in various aphid species, even using direct systems of sample preparation. This novel diagnostic could greatly simplify and reduce the cost of routine detection of this highly prevalent disease in certification and sanitary programs.It is widely assumed that fleshy fruits are involved in mediating the attraction of seed dispersal organisms and the avoidance of consumption by seed predators. It is thought that the primary function of secondary metabolites present in immature fruits is to defend them against all types of potential consumers. Changes in size, texture, taste, aroma and color occur during ripening. Frugivores include not only legitimate dispersers such as vertebrates and birds but also less appreciated but more abundant consumers of fleshy fruits, microbes. Plant volatile organic compounds comprise a wide diversity of low-molecular-weight secondary metabolites, including terpenoids. In general, flowers and fruits release the widest variety of VOCs, with emission rates peaking before pollination and at ripening. Sweet orange fruits accumulate mainly terpenoids in mature peel oil glands,vertical grow system and Dlimonene accounts for about 97% of their content. In nature, D-limonene content is usually low in orange fruits during the 2 to 3 months post-anthesis; it then drastically increases when the fruit is still green but contains seeds and remains at a high level until the fruit becomes fully mature. To investigate the role of VOCs in mature fruit interactions with specialized pathogenic microorganisms, we have generated transgenic orange plants carrying a D-limonene synthase gene in antisense configuration. Transgenic expression caused a dramatic decrease in the accumulation of D-limonene in fruit peels, being about 80-100 times lower in AS samples than in empty vector transgenic ones. A global gene expression analysis of these fruits linked the decrease of D-limonene to the upregulation of genes involved in innate immunity. Additionally, this caused the activation of J jasmonic acid signalling and metabolism upon challenge with different economically important fungal and bacterial pathogens, which led to strong general resistance against Xanthomonas citri subsp. citri, Penicillium digitatum and PhyllocThista citricarpa in AS orange peels, indicating that D-limonene and related terpene accumulation not only attract legitimate seed dispersers but also facilitate infection by specialized microorganisms.Citrus tristeza viruscauses one of the most devastating diseases of citrus worldwide inducing the death of sweet orange, mandarin, lime and grapefruit trees budded on sour orange. The availability of a CTV-resistant rootstock with the sour orange attributes of productivity, fruit quality and tolerance to abiotic stresses would be a major benefit to the citrus industry worldwide. The objective of the field trial was to evaluate the response to CTV of 10 sour orange transgenic lines carrying CTV-derived sequences. They were obtained in the laboratories of IVIA, Spain and planted at the INTA Experiment Station in Concordia, Argentina where CTV is endemic and efficiently transmitted by the brown citrus aphid .
Rooted cuttings of transgenic sour orange lines were budded with non-transgenic and virus-free Valencia Late sweet orange . Valencia trees budded on tolerant rootstocks as well as on non-transgenic sour orange were planted as controls. Trees were planted in a complete randomized design with two trees per plot and 5 replications. Every six months imprints were taken to determine the progress of CTV infection in each tree. Based on direct immuno printing-ELISA, differences in disease progress were observed till June 2012 on the different transgenic rootstocks. By December 2012 the percentage of diseased trees was over 80%. The sudden increase in disease progress in the last semester could be due to post-freeze effects. Four years after planting, almost 100 % of the trees are CTV infected, showing stunted growth and yellowing of foliage. Trees from each transgenic line were grouped according to symptom severity in the field. The better looking trees were those of two of the ten transgenic lines carrying CTVderived sequences.Citrus tristeza disease was reported in Northeast Argentina in 1930 and in the Northwest in 1947. Later, millions of citrus trees on sour orange died from quick decline in both citrus regions. The most efficient vector, Toxoptera citricida and other aphids are present and consequently, the disease is endemic. Nowadays, citrus varieties are only grafted on tolerant rootstocks. Independent of rootstock, grapefruit is affected by stem pitting, and disease expression is severe in some selections. Biological characterization of Citrus tristeza virusisolates from Northwest Argentina has been carried out since 2008 in the Centro de Saneamiento de Citrus of the EEAOC although molecular identification of isolates has not been performed thus far. In order to identify isolates, a reverse-transcription polymerase chain reaction was performed. Five sets of genotype-specific CTV primers within the open reading frame -1a of well recognized genotypes were used for characterization. CTV isolates were collected from Citrus limon, C. sinensis, C. paradisi, C. reticulata C. reshni, C. latifolia, C. macrophylla, Poncirus trifoliata and Troyer citrangeaccording to the following criteria: species or cultivars of the source tree, visual symptoms on the source tree, and symptom expression in greenhouse tests with Mexican lime , Pineapple sweet orange , sour orange and Duncan grapefruit indicator plants. Most of the source trees showed no remarkable symptomatology in the field tree. Of the five CTV genotypes analyzed, severe genotypes were widely distributed, whereas mild isolates were detected at a very low incidence. The genotypes T3 and VT were predominant in mixed infections, independent of host species and variety. Data obtained are relevant because they complement existing information for CTV biological diversity in Northwest Argentina. This is the first characterization and classification of northwestern CTV isolates.Twelve Citrus tristeza virusisolates, collected from 5 different orchards in Hunan, China, were simultaneously characterized by RT-PCR of the p23 gene, capillary electrophoresis of single strand conformation polymorphisms of the p23, p25 and p27 genes as well as multiple molecular marker analysis with four standard isolates, namely T3, T30, T36, and VT. Results from RT-PCR of the p23 gene indicated that all the isolates were virulent. CE-SSCP and MMM characterization revealed high levels of genetic diversity among the isolates ranging from single genotype infections to highly mixed infections. Out of 12 isolates, 11 contained the T3 genotype, three being single T3 genotypes, two contained T3+VT and six had T3+VT+T36+T30. The remaining isolate was the VT+T36+T30 genotype. T3 and VT, reported to be virulent genotypes, are widely distributed in Hunan Province. As the isolates were from trees showing stem pitting, the single T3 profile of CE-SSCP was likely to be related to stem pitting. Further confirmation is to be performed with more samples showing obvious stem pitting symptoms. In young orchards, single genotype isolates are usually detected, and mixed ones are found in old orchards.The characterization by the three different molecular methods resulted in consistent results with some inconsistency among different methods. In the latter case, sequencing should be conducted for further characterization.Despite millions of trees being indexed by ELISA, Citrus tristeza disease continues to spread worldwide, confirming that quarantine restrictions, eradication, and tristeza-free propagation material are not enough to combat the virus once it becomes established in an area.