An optimized duplex real-time PCR tool for sensitive detection of the quarantine oomycete Plasmopara halstedii in sunflower seeds.

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.

Development, comparison, and validation of real-time and conventional PCR tools for the detection of the fungal pathogens causing brown spot and red band needle blights of pine.

Dothistroma pini, D. septosporum, and Lecanosticta acicola are fungal pathogens that cause severe foliage diseases in conifers. All three pathogens are listed as quarantine organisms in numerous countries throughout the world and, thus, are subject to specific monitoring. Detection and identification of these pathogens still often relies on cumbersome and unsatisfactory methods that are based upon the morphological characterization of fungal fruiting bodies and conidia. In this study, we present the development of several new molecular tools that enable a rapid and specific in planta detection of each of these pathogens. Several DNA extraction procedures starting from infected needles were compared and four commercial DNA extraction kits provided DNA of satisfactory quality for amplification by polymerase chain reaction (PCR). In addition, we developed several sets of conventional PCR primers, dual-labeled probes (DLPs), and duplex-scorpion probes (DSPs), all of which targeted each pathogen. Their ability to detect the pathogens in a series of naturally infected needle samples was compared. The quadruplex DLP real-time assay proved to be more sensitive than the DSP assay and conventional PCR but the two real-time probe formats yielded identical results in the naturally infected samples. Both real-time assays proved to be significantly superior to the technique of humid chamber incubation, which often failed to produce spores for the accurate identification of the pathogens.

Sensitive detection of Fusarium circinatum in pine seed by combining an enrichment procedure with a real-time polymerase chain reaction using dual-labeled probe chemistry.

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens.

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.

Evolution of the terminal regions of the Streptomyces linear chromosome.

Comparative analysis of the Streptomyces chromosome sequences, between Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces ambofaciens ATCC23877 (whose partial sequence is released in this study), revealed a highly compartmentalized genetic organization of their genome. Indeed, despite the presence of specific genomic islands, the central part of the chromosome appears highly syntenic. In contrast, the chromosome of each species exhibits large species-specific terminal regions (from 753 to 1,393 kb), even when considering closely related species (S. ambofaciens and S. coelicolor). Interestingly, the size of the central conserved region between species decreases as the phylogenetic distance between them increases, whereas the specific terminal fraction reciprocally increases in size. Between highly syntenic central regions and species-specific chromosomal parts, there is a notable degeneration of synteny due to frequent insertions/deletions. This reveals a massive and constant genomic flux (from lateral gene transfer and DNA rearrangements) affecting the terminal contingency regions. We speculate that a gradient of recombination rate (i.e., insertion/deletion events) toward the extremities is the force driving the exclusion of essential genes from the terminal regions (i.e., chromosome compartmentalization) and generating a fast gene turnover for strong adaptation capabilities.

Differential and cross-transcriptional control of duplicated genes encoding alternative sigma factors in Streptomyces ambofaciens.

The duplicated hasR and hasL genes of Streptomyces ambofaciens encode alternative sigma factors (named sigma(B(R)) and sigma(B(L))) belonging to the sigma(B) general stress response family in Bacillus subtilis. The duplication appears to be the result of a recent event that occurred specifically in S. ambofaciens. The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level. In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent. While hasL is monocistronic, hasR is arranged in a polycistronic unit with two upstream open reading frames, arsR and prsR, that encode putative anti-anti-sigma and anti-sigma factors, respectively. Transcription of each has gene is initiated from two promoters. In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by the B. subtilis general stress response sigma factor sigma(B). Expression from this type of promoter for each of the has genes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses. Reverse transcription-PCR measurements indicate that hasR is 100 times more strongly expressed than hasL from the sigma(B)-like promoter. Transcription from the second promoter of each gene (located upstream of arsR in the case of the hasR locus) appears to be constitutive and weak. Quantitative transcriptional analysis in single and double has mutant strains revealed that sigma(B(R)) and sigma(B(L)) direct their own transcription as well as that of their duplicates. Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the sigma factors implied in stress response in Streptomyces.

End-to-end fusion of linear deleted chromosomes initiates a cycle of genome instability in Streptomyces ambofaciens.

Two mutant strains harbouring a linear chromosome whose size reached 13 Mb (versus approximately 8 Mb for the wild type) were characterized. This chromosomal structure resulted from the fusion in inverted orientation of two chromosomes partially deleted on the same arm. The fusion occurred by illegitimate recombination between 6 bp repeats. This chromosomal structure was inherited in strict association with a high level of genetic instability (30% of mutants in a single progeny, phenomenon also called hypervariability) and chromosomal instability. In contrast, derivatives, which did not retain the chromosome fusion, showed a wild-type-like instability frequency (c. 1%). Stabilization of the chromosomal structure occurred by chromosome arm replacement or circularization. A high variability of the terminal inverted repeat (TIR) length in the rescued chromosomes (from 5 kb to approximately 1.4 Mb for linear derivatives) was observed. Mutant lineages harbouring the chromosomal fusion are characterized by a highly heterogeneous distribution of DNA in the spores, by the presence of spores without DNA as well as aberrant sporulation figures, and by the production of spores with a low germination rate. The wild-type characteristics were restored in the descendants, which lost the chromosomal fusion. Thus, the fusion of deleted chromosomes initiates a cycle of chromosome instability sharing several levels of analogy with the behaviour of dicentric chromosomes in eukaryotes. We propose that the high instability of the fused chromosomes results from the duplication of a region involved in partitioning of the chromosomes (parAB-oriC ).