ARS | Publication request: Biased amplification of non-target sequences prevents PCR-based detection of plant associated microbes observed with microscopy

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 27, 2009
Publication Date: N/A

Technical Abstract: The process of amplifying rDNA from diverse microbial populations with universal primers is prone to biases that inaccurately represent population diversity. We have used a variety of fungal- and bacterial-specific primers to amplify endophyte sequences from Atriplex species and observed that 83% of the products amplified from host plant tissues using these primers exhibited pairwise alignment to plant rather than microbial genes. Such alignments can generate false impressions that these plant tissues are endophyte free. To evaluate microbial diversity in micropropagated Atriplex species, we have used a variety of PCR methods in combination with culturing, light and electron microscopy techniques. Touchdown and reconditioning PCR with primers of varying specificity failed to amplify microbes visible by microscopy. Microbes were isolated from A. canescens seeds but none of the microbes detected in micropropagated plants could be cultured. Physical separation of microbial cells may be necessary for successful amplification of novel unculturable microbes from plant tissues or other complex habitats.