HybProbes-based real-time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei, the potato common scab pathogens.

UNLABELLED: The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S. scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S. scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S. scabies and S. europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S. scabies and S. europaeiscabiei strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment.

Molecular detection and analysis of a novel metalloprotease gene of entomopathogenic Serratia marcescens strains in infected Galleria mellonella.

Serratia marcescens strains isolated from entomopathogenic nematodes (Rhabditis sp.) were examined for their pathogenicity and establishment in wax moth (Galleria mellonella) larvae. All the Serratia strains were potently pathogenic to G. mellonella larvae, leading to death within 48 h. The strains were shown to possess a metalloprotease gene encoding for a novel serralysin-like protein. Rapid establishment of the bacteria in infected larvae was confirmed by specific polymerase chain reaction (PCR) detection of a DNA fragment encoding for this protein. Detection of the viable Serratia strains in infected larvae was validated using the SYBR Green reverse transcriptase real-time PCR assay targeting the metalloprotease gene. Nucleotide sequences of the metalloprotease gene obtained in our study showed 72 single nucleotide polymorphisms (SNP) and 3 insertions compared with the metalloprotease gene of S. marcescens E-15. The metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions relative to the closest GenBank entry, S. marcescens E-15. A comparison of the amino acid composition of the new serralysin-like protein with that of the serralysin protein of S. marcescens E-15 revealed differences at 11 positions and a new aspartic acid residue. Analysis of the effect of protein variation suggests that a new aspartic acid residue resulting from nonsynonymous nucleotide mutations in the protein structure could have the most significant effect on its biological function. The new metalloprotease gene and (or) its product could have applications in plant agricultural biotechnology.

Microevolution of symbiotic Bradyrhizobium populations associated with soybeans in east North America.

Microevolution and origins of Bradyrhizobium populations associated with soybeans at two field sites (A and B, 280 km apart in Canada) with contrasting histories of inoculation was investigated using probabilistic analyses of six core (housekeeping) gene sequences. These analyses supported division of 220 isolates in five lineages corresponding either to B. japonicum groups 1 and 1a or to one of three novel lineages within the genus Bradyrhizobium. None of the isolates from site A and about 20% from site B (the only site with a recent inoculation history) were attributed to inoculation sources. The data suggest that most isolates were of indigenous origin based on sequence analysis of 148 isolates of soybean-nodulating bacteria from native legumes (Amphicarpaea bracteata and Desmodium canadense). Isolates from D. canadense clustered with B. japonicum group 1, whereas those from A. bracteata were placed in two novel lineages encountered at soybean field sites. One of these novel lineages predominated at soybean sites and exhibited a significant clonal expansion likely reflecting selection by the plant host. Homologous recombination events detected in the 35 sequence types from soybean sites had an effect on genetic diversification that was approximately equal to mutation. Interlineage transfer of core genes was infrequent and mostly attributable to gyrB that had a history of frequent recombination. Symbiotic gene sequences (nodC and nifH) of isolates from soybean sites and native legumes clustered in two lineages corresponding to B. japonicum and B. elkani with the inheritance of these genes appearing predominantly by vertical transmission. The data suggest that soybean-nodulating bacteria associated with native legumes represent a novel source of ecologically adapted bacteria for soybean inoculation.

A PCR-RFLP assay for identification and detection of Pythium myriotylum, causal agent of the cocoyam root rot disease.

AIM: Development of a PCR-RFLP assay that could reliably distinguish strains of Pythium myriotylum that are pathogenic to cocoyam from nonpathogens, as well as in planta detection of the pathogen. METHODS AND RESULTS: Sequences of the internal transcribed spacer regions of nuclear ribosomal DNA (rDNA-ITS) containing ITS1 and ITS2 of P. myriotylum isolates from cocoyam and other hosts were aligned and a restriction map was generated. rDNA-ITS alignment report revealed a new single nucleotide polymorphism (SNP; thymine/cytosine) downstream to previously published SNP (guanine/adenine) between isolates of P. myriotylum that are pathogenic to cocoyam and nonpathogenic strains. This new SNP is within the restriction site of the endonuclease AarI. Based on this SNP, a PCR-RFLP assay was developed for specific detection of P. myriotylum. The PCR amplicons of all isolates of P. myriotylum that infect cocoyam were cleaved by AarI, resulting to two bands (600/400 bp); but those from other hosts showed a single band (1000 bp), confirming the presence and specificity of the AarI restriction site. Also, the assay was effective in in planta detection of the pathogen on infected cocoyam roots without prior isolation of a pure culture. CONCLUSION: A PCR-RFLP method was developed that differentiates isolates of P. myriotylum that are pathogenic to cocoyam from nonpathogens as well as from other fungi commonly found in the cocoyam rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: Early and rapid detection of the pathogen could be of great importance in certifying planting materials as disease-free, enhancing sustainable management practices and limiting economic losses.

Miniprimer PCR assay targeting multiple genes: a new rapid and reliable tool for genotyping Pantoea stewartii subsp. stewartii.

AIM: Development of a 'miniprimer' PCR assay for genotyping Pantoea stewartii subsp. stewartii, the causal agent of the Stewart's bacterial wilt on maize. METHODS AND RESULTS: Four 10-nucleotide (10-nt) 'miniprimer' sets were designed and evaluated in the presence of Titanium Taq DNA polymerase. Under optimal reaction conditions, the miniprimer pair Uni-BacF-10/Uni-BacR-10 reproducibly generated identical banding patterns among 10 strains of P. stewartii subsp. stewartii, different patterns from strains of P. stewartii subsp. indologenes, other Panteoa species, Clavibacter michiganensis, Pectobacterium spp., Pseudomonas spp. and other bacterial species. The amplicons of Pantoea stewartii subsp. stewartii were cloned and sequenced to identify genes or DNA fragments that are targeted by the miniprimer PCR assay. Of the 14 'clone types' identified, sequences of a 1.23-kb fragment had a 99.8% similarity to part of the Pantoea stewartii zeaxanthin diglucoside biosynthetic operon (AY166713). Other dominant cloned fragments included a 411-bp amplicon that exhibited 99.8% similarity to the psaU gene (syn:ysaU; GQ249669), a type III protein-secretion system complex of P. stewartii subsp. stewartii strain DC283, and a 548-bp fragment showed 63% homology to the Asp/Glu racemase encoding gene in Erwinia tasmaniensis strain ET1/99. CONCLUSION: The miniprimer PCR assay reported here is highly discriminatory and reproducible in genotyping Pantoea stewartii subsp. stewartii. SIGNIFICANCE AND IMPACT OF THE STUDY: This miniprimer PCR assay could be a new reliable and rapid tool for fingerprinting the Stewart's wilt pathogen of maize.

Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency approximately 68 %) but was recovered infrequently ( approximately 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

Intercistronic heterogeneity of the 16S-23S rRNA spacer region among Pseudomonas strains isolated from subterranean seeds of hog peanut (Amphicarpa bracteata).

Intercistronic heterogeneity of the 16S-23S rRNA internal transcribed spacer regions (ITS1) was investigated in 29 strains of fluorescent pseudomonads isolated from subterranean seeds of Amphicarpa bracteata (hog peanut). PCR amplification of the ITS1 region generated one or two products from the strains. Sequence analysis of the amplified fragments revealed an ITS1 fragment of about 517 bp that contained genes for tRNA(Ile) and tRNA(Ala) in all 29 strains; an additional smaller ITS1 of 279 bp without tRNA features was detected in 15 of the strains. The length difference appeared to be due to deletions of several nucleotide blocks between the 70 bp and 359 bp positions of the alignment. The end of the deletions in the variant ITS1 type coincided with the start of antiterminator box A, which is homologous to box A of other bacteria. Phylogenetic analyses using the neighbour-joining algorithm revealed two major phylogenetic clusters, one for each of the ITS1 types. Using a single specific primer set and the DNA-intercalating dye SYBR Green I for real-time PCR and melting-curve analysis produced highly informative curves with one or two recognizable melting peaks that readily distinguished between the two ITS1 types in pure cultures. The assay was used to confirm the presence of the variant ITS1 type in the Pseudomonas community in total DNA from root-zone soil and seed coats of hog peanut. Heterogeneity of the ITS1 region between species has potential for studying molecular systematics and population genetics of the genus Pseudomonas, but the presence of non-identical rRNA operons within a genome may pose problems.

Evaluation of Seed and Soil Treatments with Novel Bacillus subtilis Strains for Control of Soybean Root Rot Caused by Fusarium oxysporum and F. graminearum.

Fusarium root rot is an important disease of soybean in Ontario, Canada. This study is to select antagonistic bacterial agents as effective alternatives to chemical pesticides for the control of root rots caused by Fusarium oxysporum and F. graminearum. Twenty-two Bacillus subtilis strains from soybean and corn roots were tested in dual cultures for inhibition of mycelial growth of F. oxysporum and F. graminearum. All strains significantly reduced mycelial growth of F. oxysporum by approximately 17 to 48% and of F. graminearum by 10 to 32%. Ten B. subtilis strains selected based on their larger fungal inhibition zones were evaluated against macroconidial germination. These strains inhibited the spore germination of F. oxysporum by 20 to 48% and of F. graminearum by 14 to 32% in cell-free filtrates. Under greenhouse conditions, the efficacy of seed and soil treatments with B. subtilis strains against the two Fusarium root rot pathogens was evaluated based on root rot severity, seedling emergence, plant height, and root dry weight. Six B. subtilis strains (SB01, SB04, SB23, SB24, SB28, and SB33) from soybean roots and two strains (CB01 and CH22) from corn roots significantly reduced the severity of the two Fusarium root rots in seed or soil treatments. Strains SB01, SB04, SB23, and SB24 were the most effective treatments against both pathogens in either seed or soil treatment. When applied as seed treatments, these four strains reduced root rot severity by 43 to 63% and increased emergence by 13 to 17%, plant height by 9 to 18%, and root dry weight by 8.4 to 19%. When used as soil treatments, they reduced root rot severity by 68 to 74% and increased emergence by 14 to 18%, plant height by 11 to 23%, and root dry weight by 16 to 24%. These results suggest that the novel strains of B. subtilis identified in this research can be effective alternatives to fungicides in managing Fusarium root rots of soybean, and a greater level of efficacy may be achieved when they were used as soil treatments than seed treatments.

Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene.

AIMS: The development and evaluation of a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. METHODS AND RESULTS: A TaqMan-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan PCR assay detected 1 pg of purified DNA and 10(4)P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92.0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. CONCLUSIONS: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.

Oligonucleotide array for identification and detection of pythium species.

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction.

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.