Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Abstract

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.

Citrus psorosis virus coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes

“…The mRNA-ihpCP accumulation was measured in the mother plants and their replicates. This involved analysis of the mature leaves of each ihpCP line mother plant (P 0 ), its replicates (P 1 ), and the non-tg control by RT-qPCR optimised for ihpCP transcript detection (De Francesco et al 2015). Figure 1 shows that both, the mother plant and the replicates of each line, accumulated mRNA-ihpCP at a low level compared to the ubiquitin mRNA.…”

“…To confirm the negative CPsV detection in plant samples of lines ihpCP-10 and -15, we applied a specific, highly sensitive, RT-qPCR detection protocol, as previously described (De Francesco et al 2015). Although this method also detects the ihpCP transcript present in these transgenic lines (Fig.…”

Citrus Psorosis Virus: Current Insights on a Still Poorly Understood Ophiovirus

“…Biological indexing requires one year and is currently the only and validated technique applied for citrus psorosis diagnosis [ 75 ]. In some cases, psorosis is completely symptomless in field trees over several years, and biological indexing is, therefore, an absolute requirement to verify that citrus trees are not infected with CPsV.…”

“…Although biological indexing is considered as the only validated technique for the diagnosis of citrus viruses including CPsV [ 75 ], its results remain incomplete. This is based on the fact that its sensitivity to detect CPsV varies depending on the viral isolates associated with the disease.…”

“…This test is considered as a useful rapid method that will aid in the production of virus-tested citrus plants for certification purposes [ 85 ]. Real-time RT-PCR is a well-established diagnostic method applied to many pathogens and crops, including woody plants such as citrus and its relatives [ 75 ]. Recently, many studies have shown the reliability of this tool even for the simultaneous detection of several viruses in citrus [ 75 , 86 ].…”