Comprehensive genomic analysis of the potential limitations of several published PCR primers targeting prfA-virulence gene cluster in Listeria species.

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes, foodborne pathogen. This study conducted in silico genomic analysis to investigate the specificity and binding efficacy of four published pairs of PCR primers targeting Listeria prfA-virulence gene cluster (pVGC) based on Listeria sequences available. We first performed comprehensive genomic analyses of the pVGC, the main pathogenicity island in Listeria spp. In total, 2961 prfA, 642 plcB, 629 mpl, and 1181 hlyA gene sequences were retrieved from the NCBI database. Multiple sequence alignments and phylogenetic trees were generated using unique (non-identical or not-shared) sequences of each represented genes, targeting four pairs of PCR primers published previously, namely 202 prfA, 82 plcB, 150 mpl, and 176 hlyA unique gene sequences. Only the hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB, and mpl genes showed weak (<50%) matching results. In addition, nucleotide variations were observed at the 3' end of the primers, indicating non-binding to the targets could potentially cause false-negative results. Thus, we propose designing degenerate primers or multiple PCR primers based on as many isolates as possible to minimize the false-negative risk and reach the aim of low tolerable limits of detection.

First Report of Soybean Cyst Nematode (Heterodera glycines Ichinohe) on Soybean in the Province of Quebec, Canada.

In eastern Canada, soybean, Glycine max (L.) Merr., is the most important legume, and its cultivation is expanding to new regions as cultivars for the short growing season are developed. The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is among the most destructive pests of soybean in the world. This nematode is also under quarantine regulations in many countries, including Canada. Until now, in Canada, SCN was only reported in the province of Ontario. Since its first detection in 1988 in the southwestern part of the province (1), SCN has been found in 12 other counties. It appears that SCN has been spreading in a north and northeast direction along the St. Lawrence River. We report here the first detection of SCN in the province of Quebec. Second stage juveniles (J2) and cysts were found in St. Anicet, Quebec, Canada, in a 10-ha soybean field. Light textured soil is a characteristic of the field, the same site where Pratylenchus alleni was recently discovered (2) and where irregular patches of stunted soybean plants were observed. Morphological and molecular studies of J2 and cysts confirmed the identification of this nematode population as SCN. The J2 were typical for SCN with a body length of 393 to 428 µm, lateral fields harboring four straight lines, a well-developed stylet 23 to 25 µm long, sub-ventral base knobs with posterior slops, a tail length of 43 to 50 µm, and a hyaline part of 23 to 29 µm. Cysts were brown and lemon-shaped with a posterior protuberance, ambifenestrated, underbridged, and had a strongly developed bullae. Key morphometrics were: a cyst fenestra 40 to 57 µm long and 28 to 44 µm wide, and a vulval slit 39 to 53 µm long. All of these are coincident with those of SCN (3). Ribosomal DNA of the ITS, 18S, and D2/D3 regions, and mitochondrial COX1 gene were PCR amplified from cysts and J2s gDNA using primers ITS-F (5'-TTGATTACGTCCCTGCCCTTT-3') and ITS-R (5'-ACGAGCCGAGTGATCCACCG-3'); 18S-F (5'-TTGGATAACTGTGGTTTAACTAG-3') and 18S-R (5'-ATTTCACCTCTCACGCAACA-3'); D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and D3B (5'-GACCCGTCTTGAAACACGGA-3'); and COXI-F (5'-CCTACTATGATTGGTGGTTTTGGTAATTG-3') and COX1-R (5'-GTAGCAGCAGTAAAATAAGCACG-3'), respectively, and sequenced. The nucleotide sequences were 98 to 100% similar to those of SCN found in NCBI nr database (July 2013). All the sequences have been submitted to GenBank with the following accession numbers: ITS (KF453621); 18S (KF453622); D2/D3 (KF453623); and COX1 (KF453624). Using species specific sequence characterized amplified region (SCAR) primers (4) also confirmed this was H. glycines. This is the first reported case of SCN in Quebec, Canada. The proximity of St. Anicet to the Ontario border is in accordance with the North/Northeastern dispersal hypothesis. The HG type of the SCN population will have to be determined before any resistant cultivars are deployed for the management of this pathogen in the province. References: (1) T. R. Anderson et al. Plant Dis. 72:453, 1988. (2) G. Bélair et al. Plant Dis. 97:292, 2013. (3) R. H. Mulvey. Can. J. Zool. 50:1277, 1972. (4) S. Ou et al. Nematology 10:397, 2008.

First Report of the Root-Lesion Nematode, Pratylenchus alleni Associated with Damage on Soybean in Quebec, Canada.

In eastern Canada, soybean, Glycine max (L.) Merr., is the most important cultivated legume species. In 2011, the provinces of Ontario and Quebec had 987,400 and 300,000 ha of soybean production, respectively. Root-lesion nematodes, Pratylenchus spp., are the most prevalent plant-parasitic nematodes in Canadian agroecosystems and can affect many crops (3). In 2011, irregular patches of stunted soybean plants were observed for the first time in a 10-ha soybean field grown on a light texture soil in St. Anicet, Quebec (45°4'50.51″N, 74°21'18.56″W). Yield reduction in damaged plots ranged from 38 to 54% when compared with asymptomatic adjacent plots. The field was sown with soybean cv. PRO 2715R (PRO Seeds of Canada, Woodstock, ON), a Roundup Ready cultivar of 2,750 CHU, at 100 kg/ha. Soil analysis revealed a uniform pH of 6.0 ± 0.2 on a gravelly sandy soil (81% sand, 10% loam, 9% clay, and 4% organic matter). On 7 October 2011, root samples were collected from 10 randomly selected damaged patches, washed under running water, and deposited in a mist chamber for a 14-day extraction period. Specimens were stored in tap water at 4°C before identification. Based on morphological characteristics, 26 individual specimens were examined (14 females, 11 males, one juvenile) and were all identified as Pratylenchus alleni Ferris, 1961 (2). Genomic DNA was extracted from individual larvae and the internal transcribed spacers (ITS) region was amplified. RFLP analysis with five restriction enzymes (CfoI, DdeI, HindIII, HpaII, and PstI) showed that the banding pattern for this species was different from those of 18 other major Pratylenchus species (4). Sequence of ITS regions of this population (GenBank Accession No. JX081545) confirmed genus identification but showed only limited homology (<85%) with the 20 Pratylenchus species available in the database at this time. Based on the morphological identification and the ITS sequence divergence with other important species, the sequence was deposited as the first P. alleni accession in GenBank. In the United States, the pathogenicity of P. alleni to soybean is well established (1). To our knowledge, this is the first report of P. alleni occurrence and damage to soybean in Canada. The outbreak of this root-lesion nematode will pose a new challenge for crop management since no registered compounds are currently available against this pest in soybean in Canada. References: (1) N. Acosta and R. B. Malek. J. Nematol. 13:6, 1981. (2) V. Ferris. Proc. Helminthological Soc. Washington 28:109, 1961. (3) J. W. Potter and A. W. McKeown. Can. J. Soil Sci. 83:289, 2003. (4) L. Waeyenberge et al. Nematol. 2:135, 2000.