Detection of Pasteuria penetrans infection in Meloidogyne arenaria race 1 in planta by polymerase chain reaction

• Published in: FEMS microbiology ecology 2004-06, Vol.48 (3), p.457-464
• Main Authors: Schmidt, L.M., Preston, J.F., Nong, G., Dickson, D.W., Aldrich, H.C.
• Format: Article
• Language: English
• Subjects: Animals; Bacillus subtilis - genetics; Bacteria; Bacterial Proteins - genetics; Bacteriology; Biological and medical sciences; Biological control; Cluster Analysis; Deoxyribonucleic acid; DNA; DNA Primers - genetics; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; DNA, Ribosomal - genetics; Ecology; Fluorescence; Fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; Galls; Genes; Gram-Positive Bacteria - genetics; Gram-Positive Bacteria - isolation & purification; Gram-Positive Bacterial Infections - microbiology; Gram-Positive Bacterial Infections - veterinary; Gram-positive endospore-forming bacteria; Hybridization; Lycopersicon esculentum; Lycopersicon esculentum - parasitology; Meloidogyne arenaria; Microbiology; Miscellaneous; Nematodes; Pasteuria penetrans; PCR detection; Phylogeny; Polymerase chain reaction; Polymerase Chain Reaction - methods; Race; RNA, Bacterial - genetics; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sequence Homology; sigE; Sporulation; Tomatoes; Tylenchoidea - microbiology
• ISSN: 0168-6496; 1574-6941
• DOI: 10.1016/j.femsec.2004.03.011

We report on the development of a PCR-based assay to detect Pasteuria penetrans infection of Meloidogyne arenaria in planta using specific primers for recently sequenced sigE, spoIIAB and atpF genes of P. penetrans biotype P20. Amplification of these genes in crude DNA extracts of ground tomato root galls using real-time kinetic PCR distinguished infected from uninfected M. arenaria race 1 by analysis of consensus thresholds for single copy genes. Fluorescent in situ hybridization (FISH) using the sigE primer sequence as a probe shows hybridization to P. penetrans cells in various stages of vegetative (pre-endospore) development. Ratios of gene copies for sigE and 16S rDNA were obtained for P. penetrans and compared to Bacillus subtilis as a genomic paradigm of endospore-forming bacteria. Phylogenetic analysis of the sigE gene from Gram-positive, endospore-forming bacteria finds P. penetrans most closely related Paenbacillus polymyxa. The sporulation genes ( spo genes), particularly si gE, have sequence diversity that recommends them for species and biotype differentiation of the numerous Pasteuria isolates that infect a large number of plant-parasitic nematodes.