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.

Botrytis leaf blight (BLB) of onion (Allium cepa) is caused by Botrytis squamosa. The disease has been reported on onion crops in several of the onion production areas of the world including North and South America, Europe, Asia, and Australia, although it is not a problem in arid production regions such as the western United States. In eastern Canada, the disease is generally present every year and is especially severe on cultivars of yellow globe onion. The pathogen biology and disease epidemiology have been intensively researched. Over the last few decades, in the organic soil area of Quebec, extensive research effort has been devoted to the development and evaluation of predictive models and disease management strategies. There has been an active integrated pest management program for onions since the early 1980s, and scouting for disease has played a major role in disease management. In this article, the story of BLB management in eastern Canada over a period of two decades is summarized.

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay’s specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/μl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

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.

With the pyrosequencing assay developed in this study, a large number of Botrytis spp. individuals can be characterised in a timely fashion with greater accuracy than by commonly used methods. Hence, pyrosequencing-based methods will be useful for improving our understanding of fungicide resistance, detecting the arrival of new genetic substitutions, monitoring shifts in fungal populations and assessing the effectiveness of antiresistance strategies, and for routine monitoring of fungicide resistance.