An UltraClean Microbial DNA Kit (MO BIO Laboratories,
Inc., Carlsbad, CA) was used to extract microbial genomic DNA
from seed tissues according to the manufacturer’s instructions.
DNA (10 to 15 ng/μl) was subjected to real-time PCR assay using
A. citrulli-specific primers as previously described (10). Samples
with cycle threshold (Ct) values less than 35 were considered to
be positive for A. citrulli. To ensure that there was no PCR inhibition
in samples that yielded negative results, 5 ng/μl of A.
citrulli genomic DNA was added to DNA preparations from 10
control seed samples and subjected to real-time PCR as described