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A primer‐introduced restriction analysis‐PCR‐based method to analyse Pepper mild mottle virus populations in plants and field soil with respect to virus mutations that break L 3 gene‐mediated resistance of Capsicum plants
Author(s) -
Sakamoto M.,
Tomita R.,
Hamada H.,
Iwadate Y.,
Munemura I.,
Kobayashi K.
Publication year - 2008
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/j.1365-3059.2008.01835.x
Subject(s) - biology , restriction enzyme , gene , tobamovirus , virology , polymerase chain reaction , virus , genetics , primer (cosmetics) , restriction fragment length polymorphism , plant virus , organic chemistry , chemistry
One or a few nucleotide changes in the coat protein gene reportedly confer Pepper mild mottle virus (PMMoV) with the ability to overcome L 3 gene‐mediated resistance in Capsicum plants. Primer‐introduced restriction analysis‐PCR (PIRA‐PCR) was set up to detect four known resistance‐breaking mutations. Each mutation from the L 3 ‐breaking PMMoV strains was successfully detected by reverse transcription‐PCR amplification of the viral coat protein gene, PCR amplification of the regions containing the mutations with restriction site‐introducing primers, followed by restriction analysis. Since PIRA‐PCR primers were designed such that only PCR products from L 3 ‐breaking PMMoV strains can be digested by appropriate restriction enzymes, L 3 ‐breaking strains could be detected when they comprised no more than 20% of the mixture with non‐ L 3 ‐breaking strain. Using this PIRA‐PCR‐based method, L 3 ‐breaking PMMoV was detected in field soil samples taken from the base of both diseased and non‐diseased plants harbouring L 3 . The results show that PIRA‐PCR is useful to quickly detect L 3 ‐breaking PMMoV strains not only in Capsicum plants harbouring the L 3 resistance gene but also in field soil, which serves as a reservoir of infectious viruses.