Improvement of long segment ribosomal PCR amplification for the molecular taxonomic identification of Litylenchus crenatae mccannii in beech trees with beech leaf disease

Authors: Carta, Lynn; Li, Shiguang

Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/7/2019
Publication Date: 3/17/2020
Citation: Carta, L.K., Li, S. 2020. Improvement of long segment ribosomal PCR amplification for the molecular taxonomic identification of Litylenchus crenatae mccannii in beech trees with beech leaf disease. Journal of Nematology. 52:e2020-016. https://doi.org/10.21307/jofnem-2020-016.
DOI: https://doi.org/10.21307/jofnem-2020-016

Interpretive Summary: Nematodes are microscopic worms that attack plants and cause billions of dollars of damage to crops and forest and ornamental trees. One problem in identifying nematodes for their damage potential is that the quantity and quality of the nematode sample may be poor. Besides the initial microscopic observation, more refined identifications require DNA profiling that can distinguish very closely related populations. However reliably generating these DNA marker sequences requires molecular tools and ingredients of just the right types and combinations to get the longest possible stretch of information for the most accurate identification of even poor quality specimens. Therefore ARS scientists systematically tested which chemicals can improve the reactions needed to generate the largest product from specimens that failed initial laboratory procedures to unlock their genetic information. They discovered that a combination of two special proteins called enzymes and a special salt solution could generate a product that previous recipes used on difficult samples could not. This was demonstrated using Litylenchus crenatae mccannii nematodes received from beech leaf disease samples from Ohio and Pennsylvania that are the subjects of surveys in the northeastern US. The new combination of ingredients and methods are described so that nematodes important for agricultural trade and active disease management can be more reliably identified from small amounts of DNA. This information will be used by agricultural researchers and diagnosticians in the U.S. and other countries.

Technical Abstract: Generating DNA markers for microscopic plant parasitic nematodes can be especially difficult if only a few valuable, tiny specimens are available. Providing a reliable maximum amount of unambiguous genetic information from single nematodes is especially important when identifying damaging, regulated nematodes of importance to trade where a few nucleotide differences in diagnostic markers are significant. There are many possible reasons for difficulty amplifying unpurified nematode DNA for long range PCR followed by direct sequencing. Specimen age, proofreading errors and reagent compatibility during PCR are among those problems. While unsuccessful direct amplification of difficult samples may sometimes be overcome by gene cloning, a more expensive and time-consuming process. Therefore, long segment PCR of a large 3.5 kb segment of ribosomal DNA was optimized for individual difficult-to-amplify young Litylenchus crenatae mccannii (Anguinidae) nematodes by systematically testing thermostable polymerases, proofreading enzymes and buffers. The combination of thermostable DreamTaq™, proofreading Pfu polymerase, and PicoMaxx™ buffer provided the best results. These nematodes are the subject of surveys currently active at many sites in the northeastern United States. This new, optimized PCR protocol will be useful for diagnostic labs associated with the surveys.