title: Detection and quantification of three distinct Neotyphodium lolii endophytes in Lolium perenne by real time PCR of secondary metabolite genes.
abstract: Perennial ryegrass (Lolium perenne) is a widely used pasture grass, which is frequently infected by Neotyphodium lolii endophytes that enhance grass performance but can produce alkaloids inducing toxicosis in livestock. Several selected endophyte strains with reduced livestock toxicity, but that confer insect resistance, are now in common use. Little is known regarding the survival and persistence of these endophytes when in competition with common toxic endophytes. This is mainly because there are currently no assays available to easily and reliably quantify different endophytes in pastures or in batches of seeds infected with multiple strains. We developed real time PCR assays, based on secondary metabolite genes known to differ between N. lolii endophyte strains, to quantify two selected endophytes, AR1 and AR37, and a common toxic ecotype used in New Zealand. A duplex PCR allowed assessment of endophyte:grass DNA ratios with high sensitivity, specificity and precision. Endophyte specific primers/probes could detect contamination of AR37 seeds with other endophytes down to a level of 3-25%. We demonstrated that it is possible to quantify different endophyte strains simultaneously using multiplex PCR. This method has potential applications in management of endophytes in pastures and in fundamental research into this important plant-microbe symbiosis.
authors.affiliation: Institute of Fundamental Sciences, College of Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand. Electronic address: Y.Zhou1@massey.ac.nz.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Institute of Fundamental Sciences, College of Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand.
authors.forename: Rosie E
authors.initials: RE
authors.lastname: Bradshaw
authors.affiliation: AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Johnson
authors.affiliation: AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand.
authors.forename: David E
authors.initials: DE
authors.lastname: Hume
authors.affiliation: AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Simpson
authors.affiliation: Institute of Fundamental Sciences, College of Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand.
authors.forename: Jan
authors.initials: J
authors.lastname: Schmid
mesh_terms: D015169:Colony Count, Microbial; D060026:Endophytes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008129:Lolium / Q000382:microbiology*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D055328:Neotyphodium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009520:New Zealand; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: Duplex PCR; Perennial ryegrass; Secondary metabolite genes; Seed contamination; TaqMan probe