CEVd has a wide host range and has been identified naturally infecting hosts other than citrus. CEVd is the largest among citrus viroids and has the unique property of spontaneously increasing in length its RNA genome by terminal repeats after prolonged infections on specific solanaceous hosts. Hop stunt viroidis the causal agent of the cachexia and xyloporosis disease and its name refers to the stunting effect induced in hops. HSVd belongs to the Hostuviroid genus and it has a wide host range. The HSVd variants identified in citrus have a size ranging from 296 to 301 nucleotides and only variants containing specific RNA sequences, also known as cachexia expression motif, cause cachexia disease on sensitive citrus such as mandarins and some of their hybrids. Citrus bark cracking viroidbelongs to the Cocadviroid genus and its name refers to the symptoms induced on trifoliate root stock.Citrus bent leaf viroidand Citrus dwarfing viroidbelong to the Apscaviroid genus . The CBLVd name refers to the symptom induced in ‘Etrog’ citron whereas CDVd refers to the size reduction of citrus trees propagated on trifoliate root stock. Two additional citrus viroids, also belonging to the Apscaviroid genus, have been identified since the 1980s. Citrus viroid Vinduces mild reactions in ‘Etrog citron’ however, synergism with other Apscaviroids results in enhanced citron symptoms. CVd-VI also induces mild reactions in ‘Etrog citron’ and has chimeric features related to CDVd, CEVd, and CBCVd. The effects of CVd-V and -VI on citrus under field conditions are still unknown. In the late 1990s, the term ‘transmissible small nuclear ribonucleic acid’ was introduced to describe well-characterised citrus viroid RNA species that do not induce distinct disease syndromes in most citrus hosts but rather act as regulatory genetic elements modifying tree performance to the benefit of the grower.

Since then, the TsnRNA-Ia, -IIa, and -IIIb have been studied in lengthy replicated field trials providing interesting results for reduced tree height and canopy volume,frambuesas en maceta enhanced fruit size and increased yield per canopy volume as well as achievement of high density plantings in the absence of any adverse effects in fruit quality or tree longevity. It is important to note however, that such results have been achieved only with specific scion/root stock/TsnRNA combinations /trifoliate/TsnRNA-IIIb and clementine /Carrizo citrange /TsnRNA-Ia+IIa+IIIb. TsnRNAs had no effect on various other scion root stock combinations /C. macrophylla and Oroblanco /Citremon or even on root stocks such as Carrizo citrange when used as seedlings. In the following decades, the advent of molecular biology provided a variety of new tools for viroid research and detection. Methods such as imprint and blot hybridization, reverse transcription and polymerase chain reaction followed by cloning and sequencing or single-strand conformation polymorphism and transient or transgenic expression of viroid RNA in planta, in combination with in vitro transcription and plant inoculation or protoplasts transfection and more recently deep sequencing and a real time quantitative PCR protocol for the universal detection of citrus viroids, have transformed our diagnostic capacity. Even though, ‘Etrog’ citron and sPAGE remain the golden standard for citrus viroid detection, since it can detect all viroid-like molecules regardless of available RNA sequence information, the need for the development of robust, quick, reliable and economical viroid detection methods is always current. Nowadays, the open trade agreements, the global movement of citrus germplasm and competition of citrus producers, in combination with the ever-changing quarantine regulations and the constant need for pathogen-tested citrus propagative materials, make the use of modern molecular technologies for citrus viroid detection a necessity. With continuing research much information has been generated regarding viroid replication , host processing , evolution and population structure , cell to cell and long distance movement , biologically active RNA secondary structures , and mechanisms involved in pathogenesis and symptom expression .

However, a series of interesting and challenging practical and basic science questions remain open. Are viroids associated with the gummy bark disease of sweet orange? Are viroids associated with “Wood pitting Gum pocket- Gummy pitting” observed on trifoliate root stock? Are viroids associated with the Kassala disease of grapefruit? Do modern molecular viroid detection methods need to replace bio-indexing? In the absence of true dwarfing citrus species and root stocks and in the face of serious citrus production cost and disease challenges, is the use of TsnRNAs for dwarfing and high-density plantings feasible in commercial scale and ethical? In the absence of any viroid encoded proteins, are viroids using a novel process for the suppression of the gene silencing plant antiviral mechanism? 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.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, and D limonene 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,hydroponic grow kit 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 Phylloctista 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 root stock 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 root stocks 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 root stocks. 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 CTV derived 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 root stocks. Independent of root stock, 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.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. Reports from all over the world show that several destructive isolates of CTV, not dependent on sensitive root stocks, may suddenly appear as a result of rearrangements or mutations of the genome. Also, it appears that bio-indexing on indicator plants or other official equivalent methods cannot help to limit the introduction of exotic strains, given that biological indexing is time consuming and molecular methods have a limited range of discrimination. With this in view, and thanks to the recent progress in CTV genome sequencing, we developed a fast diagnostic assay in which multiplex RT-PCR combined with a sequential hybridization step on the InCheck Platform allows the genotyping of CTV isolates. The 44 probes were designed on the complete genome of 38 CTV representative isolates of six phylogenetic clades . Tests carried out with isolates from different countries were used to validate the diagnostic procedure: single or mixed MMM genotypes inducing seedling yellows and stem pitting on sour orange and grapefruit; VT+T3 and VT genotypes inducing SY/SP on sweet orange; and mild isolates, with T30+T3 or T30 genotypes. Quadruplex primers Qua1 and Qua2 targeting eight genes, and, coupled with the panel of specific probes, after the hybridization step, resulted in signals of VT-like group probes for SY isolates and RB probes for isolates inducing SY and SP. T30-like isolates from asymptomatic as well as combined infections were also detected. According to the results the detection and hybridization process is easy, rapid and accurate and can also be run by someone with no background in biology. With such potential it could dramatically increase the capability of diagnostic laboratories and contribute to minimizing the impact of new emerging CTV strains.Unlike most citrus infecting viruses, Citrus tristeza viruspossesses a number of distinct strains that produce a range of disease syndromes on different host species.