Evidence for Differences in the Temporal Progress of Plasmopara viticola Clades riparia and aestivalis Airborne Inoculum Monitored in Vineyards in Eastern Canada Using a Specific Multiplex Quantitative PCR Assay.

Four clades of Plasmopara viticola isolated from wild and cultivated Vitis species were described in 2013. Only P. viticola clades riparia and aestivalis have been detected in eastern Canada. To increase our understanding of the epidemiology of these clades of P. viticola, airborne sporangia were monitored with spore samplers at two experimental vineyards from 2015 to 2018 and at 11, 14, and 15 commercial vineyards in 2016, 2017, and 2018, respectively. At each vineyard and in each year, airborne sporangia were assessed three times weekly from grapevine budbreak to harvest. To accurately monitor airborne inoculum, a specific and sensitive quantitative PCR assay for simultaneous quantification of P. viticola clades riparia and aestivalis was developed. At the experimental site, in the vineyard planted with the hybrid grape variety Chancellor, mostly P. viticola clade riparia was detected. In vineyards planted with multiple grape varieties, airborne sporangia of P. viticola clade riparia were prevalent at the beginning of the season, whereas P. viticola clade aestivalis was mostly detected from midseason to harvest. At the commercial sites, airborne sporangia of P. viticola clade riparia were more prevalent in 2016, whereas P. viticola clade aestivalis was more prevalent in 2017 and 2018. The only significant difference between the inoculum progress curves was the time at which 50% of the seasonal inoculum was reached, with an average for the 3 years of 100.8 and 117.9 days since 1 May for P. viticola clade riparia and clade aestivalis, respectively. When airborne sporangium concentrations were expressed as the proportion of the two clades, in general, the proportion of clade aestivalis to that of clade riparia was low at the beginning of the season and increased to reach approximately 0.9 to 1.0 at the end of the season. These results suggest that both clades of P. viticola coexist, but that clade aestivalis is predominant and that downy mildew epidemics caused by P. viticola clade riparia occur 2 to 3 weeks before those caused by clade aestivalis.

Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.

The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.

Incidence and Significance of Iprodione-Insensitive Isolates of Botrytis squamosa.

Botrytis leaf blight, caused by Botrytis squamosa, is an economically important disease of onion. The principal means of controlling the disease is by applying fungicides. Typical fungicide programs include applications of dithiocarbamates, chloronitriles, carboxamides, and dicarboximides such as iprodione (Rovral). Onion fields were surveyed in 2002, 2003, and 2004 for insensitivity to iprodione. Tests for insensitivity to iprodione were conducted on 62, 58, and 60 monoconidial field isolates using the automated quantitative (AQ) method with a discriminatory dose of 1.78 ppm of iprodione active ingredient (a.i.) in 2002, 2003, and 2004, respectively. Overall, insensitive isolates were detected in 51% of the fields, and the proportions of insensitive isolates were 8.1, 20.7, and 18.3% in 2002, 2003, and 2004, respectively. The aggressiveness of 10 insensitive and 18 sensitive isolates and the efficacy of iprodione was tested in planta. Onion leaves were inoculated with 750 µl of a conidial suspension of 75,000 conidia per ml and incubated in a growth chamber at 15°C. Aggressiveness was measured as lesion density (average number of lesions per cm2 of onion leaf). Lesion density varied from 2.82 to 8.04 lesions per cm2 of leaf. There was a significant effect (P < 0.0001) of isolates on lesion density. However, there was no significant correlation between lesion density and sensitivity to iprodione (r = 0.08). When onion leaves were sprayed with 1,875, 3,750, and 7,500 ppm of iprodione, percent inhibition of lesion density was higher for sensitive isolates with means of 43.04, 61.42, and 74.59, respectively. Accordingly, percent inhibition was lower for insensitive isolates with means of 13.81, 28.26, and 44.37 for iprodione concentrations of 1,875, 3,750, and 7,500, respectively. It was concluded that the incidence of insensitive isolates was relatively low, but insensitive isolates were capable of infecting onion leaves. There was a good relationship between insensitivity to iprodione in B. squamosa populations measured in vitro with the AQ method, and the reduced efficacy of iprodione in controlling Botrytis leaf blight.

Sensitivity of Botrytis squamosa to Different Classes of Fungicides.

An automated quantitative (AQ) assay was compared with radial growth on solid media and with dry weight in liquid culture for assaying fungicide sensitivity in Botrytis squamosa, the causal agent of onion leaf blight. Five isolates of B. squamosa were assayed for sensitivity to mancozeb (Dithane DG) and iprodione (Rovral) at five concentrations (0.5, 1.0, 5.0, 10.0, and 50 ppm). For mancozeb, the correlations between 50% effective concentration (EC50) values obtained with the three assays were not significant; however for iprodione, correlations between EC50 values for AQ and radial growth and for AQ and dry weight were significant (r = 0.98 and 0.99, respectively). The AQ method was less time consuming and more reliable than the two standard assays. The AQ method was used to evaluate the sensitivity of 35 field isolates of B. squamosa to mancozeb (Dithane DG), iprodione (Rovral), vinclozolin (Ronilan DF), and chlorothalonil (Bravo 500). All isolates were sensitive to mancozeb (EC50 ranged from 3.36 to 12.97) and chlorothalonil (EC50 < 1.5 µg/ml), but four isolates were insensitive to both iprodione (EC50 ≥ 3.98 µg/ml) and vinclozolin (EC50 ≥ 17.49 µg/ml). The ratio of the EC50 values of the least-sensitive and the most-sensitive isolates of B. squamosa was 1.08, 3.86, 6.98, and 37.59 for chlorothalonil, mancozeb, iprodione, and vinclozolin, respectively. Cross-resistance was observed for the two dicarboximide fungicides, iprodione and vinclozolin, with a significant correlation (r = 0.94) in the sensitivity of the 35 isolates to these two fungicides.

Complete genomic sequence of the lytic bacteriophage DT1 of Streptococcus thermophilus.

Streptococcus thermophilus lytic bacteriophage DT1, isolated from a mozzarella whey, was characterized at the microbiological and molecular levels. Phage DT1 had an isometric head of 60 nm and a noncontractile tail of 260 x 8 nm, two major structural proteins of 26 and 32 kDa, and a linear double-stranded DNA genome with cohesive ends at its extremities. The host range of phage DT1 was limited to 5 of the 21 S. thermophilus strains tested. Using S. thermophilus SMQ-301 as a host, phage DT1 had a burst size of 276 +/- 36 and a latent period of 25 min. The genome of phage DT1 contained 34,820 bp with a GC content of 39.1%. Forty-six open reading frames (ORFs) of more than 40 codons were found and putative functions were assigned to 20 ORFs, mostly in the late region of phage DT1. Comparative genomic analysis of DT1 with the completely sequenced S. thermophilus temperate phage O1205 revealed two large homologous regions interspersed by two heterologous segments. The homologous regions consisted of the early replication genes, the late morphogenesis genes, and the lysis cassette. The divergent segments contained the DNA packaging machinery, the major structural proteins, and remnants of a lysogeny module.