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

Abstract

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 sigE, 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.

Identification of New Single Nucleotide Polymorphism-Based Markers for Inter- and Intraspecies Discrimination of Obligate Bacterial Parasites (Pasteuria spp.) of Invertebrates

“…from around the globe, but there is very little understanding of the genetic variation between and within populations. Previous studies have focused on the position of P. penetrans in the bacterial kingdom (9,31,34) and the use of consensus 16S rRNA gene sequences (6,7,18) to differentiate between species and populations of Pasteuria (5,16,33). However, this information has not been extensively supported with sequence data from protein-encoding genes, which potentially provide an additional source of discrimination, with the exception of one study examining partial coding sequences of some genes in P. ramosa populations (32), as well as a recent study of genes encoding collagen-like proteins in this species (22a).…”

Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans

“…All chemicals and reagents used were reagent grade, enzyme grade, or molecular grade. P. penetrans vegetative cells were harvested from 14-to 21-day-old M. arenaria race 1-infected plants as described previously (28). Vegetative cells obtained from live 2-week-old P. ramosa P1-infected D. magna were processed as described previously (27) and placed in 50 l of 0.01 M Tris-HCl (pH 7.0)-0.01 M EDTA-0.15 M NaCl (TNE buffer).…”

“…A protocol for performing fluorescence in situ hybridization (FISH) was developed from methods described by Amann et al (1); this protocol has been described previously (28). A 5Ј-fluorescein-conjugated DNA probe, 5Ј-fluorescein-CTACGAACAGCCCTATCTATG corresponding to a segment of the P. ramosa sigE gene, was added to 20 l of hybridization buffer (20 mm Tris-HCl [pH 7.2], 0.90 M NaCl, 0.01% SDS, 20% formamide) and applied to coverslips.…”

“…and P. ramosa are based on 16S rRNA sequences, morphological properties of mature endospores, and host preferences (2,4,14,16,19,24). The phylogenetic relationships based on highly conserved sporulation transcription factors (24,28,31) and multiple genetic loci (9) further define the position of P. penetrans in relation to genomically defined Bacillus spp. All of these characteristics indicate that P. ramosa is the most phylogenetically distinct species for which comparisons in this genus have been made.…”

Pasteuria penetrans and Its Parasitic Interaction with Plant Parasitic Nematodes

“…Using a fragment of sigE Schmidt et al (2004) aligned P. penetrans closely with the genera Clostridium and Bacillus, in particular, with Paenibacillus polymyxa. To date, the only full Pasteuria gene that has been cloned and expressed was that reported by Trotter and Bishop (2003).…”