Comparison of multiple real-time PCR & real-time LAMP detection methods for the plant pathogen 'Candidatus Liberibacter' spp. causing the Huanglongbing disease on Citrus spp. (HLBVALID)

The Huanglongbing (HLB / Citrus greening) disease is associated with three species of phloem restricted bacteria recognized as members of ‘Candidatus Liberibacter’, namely ‘asiaticus’ (CLas), ‘americanus’ (CLam), and ‘africanus’ (CLaf). Although the causal agents of the disease have not been cultivated in axenic medium yet, they are inducing one of the most destructive and wide-spread disease of citrus across Asia, Africa, and America, but it was not identified in the EU or proximities. It is mainly affecting Citrus species, cultivars and hybrids and some other hosts within the Rutaceae family. This disease is associated with two main psyllids: Diaphorina citri and Trioza erytreae, and is also transmitted by propagative plant material, which can spread outbreaks on great distances. As part of an initial diagnosis, the visual inspection of symptomatic plants is a routine method for the surveillance of HLB disease, but symptoms can be misinterpreted. Yellow shoots, leaf blotchy mottle, and lopsided fruits with colour inversion and aborted seeds are typical symptoms on HLB affected trees. However, symptoms alone are not enough to complete a diagnosis, as they can be confused with nutritional disorders (zinc, iron, manganese deficiencies) or with other diseases (Citrus tristeza virus, Stubborn, Citrus blight, Australian citrus dieback). The three species responsible for the HLB can also be present in the host plant at a very low concentration and as the disease develops irregularly, individual trees may show a mixture of normal and diseased parts; symptoms can appear up to 20 months after infection. Although conventional PCR is a sensitive and specific method, the PCR tests can lead to false negative results due to the low titer and uneven distribution of the bacterium in the host plant, especially at the early stage of the infection (Jagoureix et al., 1994). Hence, conventional PCR method is not recommended for the detection of Ca. L. spp. responsible for the HLB disease in symptomless plants (Li et al., 2006), where bacterial use to be in low concentrations and in non-uniform distribution. Being more sensitive, real-time PCR tests may be useful in programs for the production of certified citrus nursery trees and in post-entry quarantine and are more adapted for early detection. Various diagnostic real-time PCR tests were published in recent years and assessed for their performance, but with different procedures. Comparison of these protocols through the same procedure is hence required in order to fully compare their performance. The three main real-time PCR tests published that are routinely used are those from Bertolini et al. (2014); Li W. et al. (2006); and Morgan et al. (2012). Proper HLB detection and identification is extremely important in preventing the entry of this disease in an area or in order to control the disease where it is already present. The recent (2015) suspicious case of HLB declared by the Portuguese authorities and the absence of confirmation, underlines the need for a thorough and comparable assessment of detection tests to guarantee the reliability of the results obtained. Therefore, collaboration at an international level would be beneficial to compare the real-time PCR protocols for the detection of Ca. L. spp. responsible for the HLB disease in Citrus spp.Report of the Euphresco project 2016-A-232 'Comparison of multiple real-time PCR & real-time LAMP detection methods for the plant pathogen 'Candidatus Liberibacter' spp. causing the Huanglongbing disease on Citrus spp. (HLBVALID)