PCR Detection of Ustilago nuda in Barley Seed
Loose smut of barley (Ustilago nuda), although not a major pathogen in the UK, can cause significant loss of yield in infected crops and can spread rapidly to uninfected plants once the spores produced on the smutted heads are released. The wind-blown spores invade healthy barley florets where they eventually produce a dormant mycelium in the embryo of the developing grain.
For seed certification purposes, the incidence of Ustilago infection in barley seed needs to be detected to a limit of 0.2%. By current methods this is achieved by extracting, staining and examining 1000 embryos per sample; a process that is extremely time consuming and costly. Seed merchants tend to test only a proportion of lots, but nearly all seed receives a prophylactic treatment to control the disease whether it is present or not. In order to reduce prophylactic use and target treatments where needed, there is a need for a sensitive, rapid detection and quantification method in order to increase the throughput of samples, and reduce the costs of testing.
Results
Primers Uh 1 and Uh 4 can amplify the Ustilago-specific DNA extracted from a highly infected (68%) seed lot. Amplification was still possible after diluting the DNA 1 in 2000, suggesting that levels of infection below 0.1% should be detectable.
The threshold of detection from seed with low levels of infection using the bulk seed extraction method was around 1%.
In order to investigate whether DNA could be extracted from embryos rather than whole seeds, which would significantly reduce the bulk of the material in the extraction process, DNA was isolated from individual embryos of a 58% infected seed lot at various stages of preparation of a standard embryo extraction procedure. As expected, around half of the embryos were positive up to stage 3 of preparation but none after stage 4.
Conclusions
Ustilago infection of barley seed can be readily detected by PCR using primers Uh 1 and Uh 4 when the seed has a high level of infection. However, a better DNA extraction method is needed in order to detect the required limit of 0.2%. This may be achieved by extracting the DNA from embryos rather than whole seed, combined with dilution of the DNA to reduce possible inhibitors in the extraction.
Primers Uh 1 and Uh 4 can amplify the Ustilago-specific DNA extracted from a highly infected (68%) seed lot. Amplification was still possible after diluting the DNA 1 in 2000, suggesting that levels of infection below 0.1% should be detectable.
The threshold of detection from seed with low levels of infection using the bulk seed extraction method was around 1%.
In order to investigate whether DNA could be extracted from embryos rather than whole seeds, which would significantly reduce the bulk of the material in the extraction process, DNA was isolated from individual embryos of a 58% infected seed lot at various stages of preparation of a standard embryo extraction procedure. As expected, around half of the embryos were positive up to stage 3 of preparation but none after stage 4.
Conclusions
Ustilago infection of barley seed can be readily detected by PCR using primers Uh 1 and Uh 4 when the seed has a high level of infection. However, a better DNA extraction method is needed in order to detect the required limit of 0.2%. This may be achieved by extracting the DNA from embryos rather than whole seed, combined with dilution of the DNA to reduce possible inhibitors in the extraction.