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ARS Home » Midwest Area » Urbana, Illinois » Soybean/maize Germplasm, Pathology, and Genetics Research » Research » Publications at this Location » Publication #146773
Title: A SIMPLE METHOD FOR PCR-AMPLIFICATION, CLONING, AND SEQUENCING OF PASTEURIA 16S RDNA FROM SMALL NUMBERS OF ENDOSPORES

Author
item ATIBALENTJA, NDEME - UNIV OF ILLINOIS
item Noel, Gregory
item CIANCIO, AURELIO - INST. OF PLANT PROT.

Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2003
Publication Date: 1/25/2004
Citation: Atibalentja, N., Neol, G.R., Ciancio, A. 2004. A simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores. Journal of Nematology. 36:100-105.

Interpretive Summary: Soybean cyst nematode (SCN) is an important yield limiting pest of soybean in all production areas of the US. Estimated annual crop loss ranges from $240 million to $1.4 billion. One area of research to control SCN that has received limited effort is biological control. The bacterium Pasteuria is an obligate parasite of nematodes, which means that it can only grow on the nematode host in soil. Species of Pasteuria have been shown to cause significant reductions in nematode numbers. The taxonomy of these bacteria has been difficult and is in a state of confusion because of the difficulty of working with an organism that is an obligate parasite of an obligate parasite (SCN on soybean). Taxonomy and classification of bacteria now requires study of the bacterial DNA. With Pasteuria this has been extremely difficult because of contamination with DNA from other bacteria in soil. We have developed a novel, more simple method of isolating and increasing DNA of Pasteuria that will allow significant progress in determining the species of Pasteuria in the myriad of 300+ Pasteuria/nematode parasitic interactions This will greatly advance knowledge of biological control of nematodes with the bacterium Pasteuria.

Technical Abstract: For many years, the taxonomy of the genus Pasteuria has been marred with confusion due to the fact that the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. Comparative analysis of 16S rDNA sequences has been used to clarify the situation at the genus level by showing that Pasteuria is member of the Alicyclobacillaceae rather than of the Actinomycetales. However, the technique depends on the availability of reliable sequence data, the production of which has been hampered by the requirement for large numbers of endospores for extraction of uncontaminated DNA needed for PCR-amplification of Pasteuria 16S rDNA. Although a major breakthrough toward in vitro cultivation of P. penetrans was announced recently, the detailed protocol is still proprietary information that may not be available to the public in the foreseeable future. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 × 106 endospores ml-1 were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae, only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of P. usgae are needed for PCR-amplification of Pasteuria 16S rDNA.