Use of a real‐time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley

Abstract

A study to investigate the association of phytoplasmas with papaya dieback and citrus decline syndromes in Ethiopia was carried out between July 2009 and February 2010, with sampling performed in major papaya-and citrus-growing areas of the Rift Valley. Samples of plants with symptoms were collected from papaya, citrus and suspected phytoplasma weed hosts and crops in and around the papaya and citrus fields studied. Nested polymerase chain reaction (nested-PCR) was used for initial characterization, using primers that amplify regions of the 16S rRNA and secA genes, and results were then confirmed with rapid real-time group-specific LAMP (loop-mediated isothermal amplification) assays. The results identified the occurrence of a phytoplasma belonging to the stolbur (16SrXII-A) group in papaya plants showing dieback symptoms, whilst no phytoplasmas were found associated with citrus decline. These results contradict previous reports that a 16SrII phytoplasma was associated with both papaya dieback and citrus decline in Ethiopia, but correspond with the association of a 16SrXII phytoplasmas with Nivum-Haamir-Dieback of papaya in Israel and papaya dieback in Australia. No 16SrXII phytoplasmas were found in any of the weeds and potential alternative hosts studied, although 16SrII phytoplasmas were consistently found in Parthenium hysterophorus weed plants. These results indicate that a 16SrXII phytoplasma is associated with papaya dieback in Ethiopia, whilst the causal agent of citrus decline is not a phytoplasma and remains unidentified.

Plant viruses in aqueous environment – Survival, water mediated transmission and detection

“…The next steps should be the design of user-friendly, on-site devices for CIM-based concentration and for molecular detection (Gutierrez-Aguirre et al, 2011). Isothermal amplification procedures, such as loop-mediated isothermal amplification (LAMP) (James et al, 2010;Nemoto et al, 2010;Tsutsumi et al, 2010;Bekele et al, 2011) enable target amplification without the need for expensive thermocyclers. In addition, LAMP amplification products can be easily detected in a lateral flow device (James et al, 2010), simplifying the methodology even more, since LAMP is a rapid and simple technique that is less prone to inhibitors present in the sample (Bekele et al, 2011).…”

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

“…LAMP is a highly specific and rapid technique, and it also circumvents the sensitivity of PCR and qPCR to inhibitors in plant extracts (Francois et al 2011); furthermore, its isothermal nature provides the potential for it to be deployed in the field (Kogovšek et al 2015;Tomlinson et al 2010a). LAMP has shown a comparable or better performance to other detection methods and a wide applicability for the detection of plant pathogenic bacteria (Lenarčič et al 2014), viroids (Lenarčič et al 2013), fungi (Tomlinson et al 2010b) and phytoplasmas (Bekele et al 2011;Dickinson 2015;Hodgetts et al 2011;Kogovšek et al 2015;Tomlinson et al 2010a).…”

Rapid and quantitative detection of Pythium inflatum by real-time fluorescence loop-mediated isothermal amplification assay

“…The generation of LAMP products can be monitored in real-time by measuring the increase in turbidity derived from magnesium pyrophosphate formation to determine the increase in amplified DNA concentration, thereby allowing quantitative detection of the target (Mori et al 2001(Mori et al , 2004Tomlinson et al 2010;Bekele et al 2011). The detection of LAMP products through fluorescence dye with an ESE-Quant tube scanner was developed; this method does not require expensive equipment or reagents and is relatively simpler and more cost effective than other DNA-based tests (Lucchi et al 2010;Njiru et al 2012;Zhang et al 2014).…”