Molecular Detection and Prevalence of Citrus Viroids in Texas

Abstract

<jats:p>Viroids are graft- or mechanically transmissible agents, disseminated through budding. Biological indexing of commercially important citrus cultivars grown in the Lower Rio Grande Valley of Texas showed that many are infected with citrus viroids. Most of these trees carried more than one viroid. In most cases, the infected trees are asymptomatic carriers because sour orange, the predominant rootstock used in Texas, does not show symptoms of viroid infection. Detection of viroids through biological indexing on sensitive indicator plants followed by sequential polyacrylamide gel electrophoresis (sPAGE) is the gold standard but is time-consuming and requires plants to be kept at optimum conditions. A conditional use of reverse transcriptase–polymerase chain reaction (RT-PCR) provides an efficient and alternative detection of viroids for use in the Texas virus-free citrus budwood certification program. RT-PCR could be useful in Texas to help expedite the evaluation for the presence of viroids before conducting the final biologic indexing. Using RT-PCR, we could detect, clone, and sequence full-length viroids of <jats:italic>Citrus exocortis viroid</jats:italic> (CEVd), <jats:italic>Hop stunt viroid</jats:italic> (HSVd) (both cachexia and noncachexia variants), <jats:italic>Citrus viroid</jats:italic>-<jats:italic>III</jats:italic> (Citrus dwarfing viroid), and <jats:italic>Citrus viroid</jats:italic>-<jats:italic>IV</jats:italic> (Citrus bark cracking viroid) from a collection of viroid-inoculated grapefruit plants. The source plants were previously shown to be viroid-infected by biological indexing on Etrog citron plants. Based on our results, RT-PCR can be a conditional substitute for biological indexing of mother trees in foundation blocks and shoot tip-grafted trees in the virus-free budwood program. A positive RT-PCR result has a serendipitous value because those trees can be discarded from the pool before expensive biological indexing.</jats:p>

Deep‐sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop

“…The viroid CBCVd is considered to be part of the citrus viroid (CVd) complexes, known to cause two very important diseases, exocortis and cachexia, although they are also associated with many other described citrus diseases (Kunta et al, 2007). Bark cracking in trifoliate orange was found to be associated with the presence of citrus viroid IV (CVd-IV) (Verniere et al, 2006).…”

“…According to reports, the main hosts of CBCVd are citrus species. Some authors describe it as a viroid of limited occurrence in nature, even in citrus orchards (Semancik & Vidalakis, 2005): in Texas it was detected in three out of nine grapefruit plants with symptoms (Kunta et al, 2007); in Greece its occurrence was recently reported for the first time and confirmed in five out of 35 citrus species and cultivars (Wang et al, 2013); while in Sudan it was present in all 24 analysed Citrus samples, regardless of the observed symptoms (Mohamed et al, 2009). It has never been discovered outside the genus Citrus, although artificial inoculation data suggest that it can be transmitted, replicate and cause a range of symptoms in some other hosts such as gynura, tomato, cucumber, chrysanthemum, aubergine, datura (Semancik & Vidalakis, 2005) and in the wild cucurbit Benincasa hispida (Puchta et al, 1991).…”

Diagnostic techniques for viroids

“…Several factors may cause decreased sensitivity of the RT‐PCR, including inhibitors (polysaccharides, secondary metabolites, polyphenols) and inefficient synthesis of viroid full‐length cDNA during the reaction (Bernad & Duran‐Vila, ; Kunta et al ., ). These problems can be avoided using modified extraction procedures that give highly purified viroids, are able to remove inhibitors, or are milder and release only a few inhibitors, in addition to optimization of primer design and amplification conditions (Shamloul & Hadidi, ; Kunta et al ., ). Nucleic acid extraction procedures have been a limitation of RT‐PCR, because they are complicated and time‐consuming.…”

“…Rapid, reliable, sensitive and specific amplification from trace amounts in a complex mixture of templates can only be achieved if the target sequence is known and primers can be designed. The sample size required for reaction is reduced to 1-100 pg total nucleic acids from infected tissue (Narayanasamy, 2011a Several factors may cause decreased sensitivity of the RT-PCR, including inhibitors (polysaccharides, secondary metabolites, polyphenols) and inefficient synthesis of viroid full-length cDNA during the reaction (Bernad & Duran-Vila, 2006;Kunta et al, 2007). These problems can be avoided using modified extraction procedures that give highly purified viroids, are able to remove inhibitors, or are milder and release only a few inhibitors, in addition to optimization of primer design and amplification conditions (Shamloul & Hadidi, 1999;Kunta et al, 2007).…”

Biological assays for plant viruses and other graft‐transmissible pathogens diagnoses: a review

“…It can be affected by interactions among different disease agents and Vidalakis et al (2004) have demonstrated that mixed infections can severely reduce the efficacy of diagnosis. Moreover, a diagnosis based on symptom expression only can also mask mixed infections, which are later revealed by serological or molecular tests (Singh & Ready, 2003;Kunta et al, 2007;Thakur et al, 2011). Even if no symptoms are observed it is recommended that further tests should be done, using for example, serological or molecular tests.…”