Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Phoma macdonaldii, the Causal Agent of Sunflower Black Stem

Abstract

Phoma macdonaldii, the causal agent of sunflower black stem, severely affects sunflower yield and quality. A rapid and sensitive detection method is necessary for diagnosis of this disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the pathogen from diseased sunflower tissues. The LAMP primers were designed targeting the rDNA region of the fungus. The reaction condition was optimized to 60°C water baths for 45 min. The detection limit of the LAMP assay was 100 fg DNA or 10 conidia/g seeds. The LAMP assay was validated by detecting P. macdonaldii from infected sunflower tissue samples including leaves, stems and seeds, and applied to seed samples randomly collected from sunflower fields. This LAMP assay will be useful to estimate disease prevalence and implement sustainable management of sunflower black stem.

Development of Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Pyrenophora graminea in Barley Seeds

“…For the control, the samples were also tested with specific SCAR primers PG-2 F (5 -CTTCTTAGCTGGGGCTACCGTC-3 ) and PG-2 R (5 -ACCGACTCGGGAAAAGAGCA-3 ) for the identification of P. graminea [22]. The PCR reaction (25 µL) consisted of 2.5 µL of 10× buffer, 2 mM MgCl 2 solution, 0.5 µM each of PG-2 F and PG-2 R primers, 0.2 µM dNTPs, and 1 U Taq DNA polymerase.…”

“…The current crop and seed health tests include morphological observation of pathogen colonies after incubating barley seeds on blotter or agar plates [13,14,19] and/or detection of pathogen-specific DNA fragments based on the conventional polymerase chain reaction (PCR) or real-time PCR [20][21][22]. The morphological identification requires a skillful technique to distinguish P. graminea from other fungi.…”

“…In 2001, Taylor et al developed a P. graminea-specific SCAR (sequence characterization amplification polymorphism) primer set, PG-2 F/R, which has been used to identify P. graminea from barley [8,23]. PCR-based tests significantly reduce the time, but they demand relatively expensive thermocyclers and imaging systems [8,20,22]. In addition, the above experimental methods need to be operated using professional instruments in the laboratory for disease identification, but the LAMP assay can meet the requirements of rapid disease detection in the field.…”

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of the potato wart pathogen Synchytrium endobioticum

“…LAMP diagnostic protocols have been developed for different plant diseases (Huang et al 2017; Sarkes et al 2020; Yang et al 2021; Sun et al 2022). However, it has not been applied on the detection of S. endobioticum .…”

“…The results of LAMP can be visually detected and therefore LAMP can be applied with portable equipment. LAMP diagnostic protocols have been developed for different plant diseases (Huang et al 2017;Sarkes et al 2020;Yang et al 2021;Sun et al 2022). However, it has not been applied on the detection of S. endobioticum.…”