Differences in personal success rate may be because of the amount of regular practice, critical evaluation to determine the cause of success or failure, sharp tools, and attention to the most minute of details.The practice of shoot-tip grafting to eliminate viroids and deleterious pathogens has been very beneficial to the citrus industry world-wide over recent decades. This not only allowed for the safe movement of new varieties around the world but also the use of quality-inducing root stocks like citranges. It has also extended the productive life of citrus orchards and in some cases improved the horticultural performance of cultivars and thereby economic value to farmers. However, the single minded goal of removing all possible pathogens from the plant material seems to have occasional deleterious effects on the horticultural performance of some cultivars. In practice, new citrus varieties are usually discovered via chance mutation in commercial orchards. Such varieties result from old clone material, usually containing one or more viruses/viroids, necessitating clean-up, as specified by relevant Plant Improvement Schemes prior to commercial release. However, the ever-improving science of virus detection and elimination makes the achievement of totally sterile plant material a reality. It is suggested that this influences the horticultural expression of such pure varieties. The authors will present information aimed at demonstrating that complete virus/viroid elimination may satisfy the regulatory hurdles for commercial release but also in some cases result in the loss of economic value. Financial return to the orchard is dependent on a few very basic aspects: early production of good yields of edible fruit with high pack-outs. Altering one or more of these characteristics may mean the difference between a winner and a loser. Beneficial and/or relatively non-harmful aspects of specific viroids/pathogens to horticultural value in fruit trees/horticulture will be outlined.
An argument will be put forward for the characterization of certain citrus viroids relative to their role in horticultural expression and the potential reintroduction of purified strains after the clean-up process to ensure maintenance of such economic value. This presentation aims at stimulating debate and is not intended to criticize or lay blame.There is a large diversity of viruses and viroids that infect citrus trees,stacking pots some of them well characterized and known, belonging to the genera Closterovirus, Ilarvirus, Capillovirus, Sadwavirus, Mandarivirus, Marafivirus, Spirovirus , Badnavirus and viroids belonging to the genera Apscaviroid, Cocadviroid, Hostuviroid and Pospiviroid. In addition, there are a large number of well-known graft transmissible diseases of unknown etiology, which suggests that there exist unknown viral agents infecting citrus. Although the diagnosis of known agents is straightforward by serological or molecular methods, the detection of viruses without prior knowledge remains a challenge. Currently, only biological indexing is able to address the detection of the majority of graft-transmissible diseases. However, the development of next generation sequencing technologies allows exceptional sequence data generation at a fraction of the cost of biological indexing, dramatically modifying the diagnostic landscape. Virus-derived small interfering RNAs from citrus trees showing different symptomatologies were analyzed by Illumina sequencing. De novo contigs generated by different bio-informatic software were analyzed against the Genbank database, using Blastn, Blastx and Tblastx. Those nucleotide sequences, which had some homology to viral sequences, were used along with all siRNAs to reconstruct the genomes of various viral agents through iterative mapping against contigs. Along with known viruses and viroids, new citrus Bunyavirus, Badnavirus and Luteoviridae species were successfully identified. NGS is a powerful technology that could greatly simplify the screening, routine diagnosis, detection and characterization of citrus pathogens, providing knowledge to generate new diagnostic tools and having the potential to rapidly replace biological indexing.Citrus stubborn disease , caused by the bacterial pathogen Spiroplasma citri, is of major concern for citrus production around the world. The development of a reliable virulence assay for S. citri is urgently needed, but has been proven to be extremely challenging. With the current project we investigated the mechanical inoculation of plants using an S. citri axenic culture. A secondary culture of S. citri isolate C189 was injected with a vaccination gun or slashed into the stems of citrus and periwinkle seedlings.
The progression of the infection was monitored using a newly developed TaqMan quantitative polymerase chain reaction assay targeting the S. citri spiralin gene, as well as an antibody ScCCPP1 detecting a secreted protein of S. citri . Our results indicated that S. citri enThered the phloem tissues and established infection in both citrus and periwinkle. Bacterial cells could be detected by qPCR in plant tissues from newly emerged tissues away from the original inoculation site 1-7 weeks post inoculation. The ScCCPP1 antibody verified the qPCR results by producing positive reactions for the same time frame. S. citri detection past 5-7 weeks post mechanical inoculation became erratic by qPCR while the ScCCPP1 antibody continued to produce positive results. These data indicate that S. citri may have failed to maintain a long term infection in these plants while S. citri secreted proteins were still present in the inoculated plants. This is the first record of successful or at least partially successful mechanical transmission of S. citri from axenic culture directly into host plants. Further improvement on the inoculation procedures to achieve long term infection and overcome the possible loss of S. citri pathogenicity due to in vitro subcultures is underway. Viroids are small, non-coding, non-encapsidated, single-stranded, covalently closed RNAs that replicate autonomously when inoculated in their plant hosts in which they may elicit diseases. Presently, more than thirty viroids have been described and classified into two families, Pospiviroidae and Avsunviroidae. In the case of citrus, the viroid journey began in 1934 with the report on the xyloporosis disorder of Palestine sweet lime followed by the reports on the cachexia disease of Orlando tangelo and the exocorThis disorder of trifoliate orange root stock in 1948. The etiological agents of these diseases, that were known to be graft-transmissible, were considered to be viruses until viroids were described as a new class of plant pathogens with the discovery of the Potato spindle tuber viroidand the Citrus exocorThis viroidin the early 1970s. CEVd was characterized after its transmission to gynura . However, since nucleic acid technologies were limited in the 1970s, ongoing studies relied on the use of experimental herbaceous hosts displaying viroid symptoms and yielding high viroid titres. This approach prevented the identification of other citrus viroids with narrower host ranges until the following decade. Following the adoption of ‘Etrog’ citron as an indicator for biological indexing of exocor.This, in the late 1970s, the range of mild to moderate and severe symptoms observed after graftinoculation with field isolates were erroneously considered as evidences for the existence of CEVd strains.
In the mid-1980s, and after the use of a double gel electrophoresis system and silver staining,grow lights four additional viroid-like RNAs with distinct electrophoretic mobilities were identified and assigned the Latin numerals I through IV. Citrus viroidI, -II, -III, and -IV were further characterized and shown to have distinct biological and molecular properties, thus considered individual viroid species separate from CEVd. During the following two decades there was much discussion regarding the names adopted for citrus viroids. Presently, after lengthy field characterization experiments and according to the criteria of the International Committee of Virus Taxonomy, the following has been accepted for the five citrus viroids characterized in the mid-1980s. All identified citrus viroids belong to genera of the Pospiviroidae family and share some structural and replication properties. CEVd is the causal agent of the exocorThis disease and its name refers to the exocorThis disease as originally described for the trifoliate root stock. CEVd belongs to the Pospiviroid genus . The available data shows that differences in virulence are host dependent and associated with certain nucleotide changes located in a specific region of the viroid RNA.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 rootstock. CBCVd is the smallest of the known citrus viroids and is closely related to CEVd. 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 rootstock. 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 inTheresting results for reduced tree height and canopy volume, 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/rootstock/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 rootstock? 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 rootstocks 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?