A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pathovar (pv.) undulosa. The protocol can also detect X. translucens pv. translucens and X. translucens pv. secalis, but can’t differentiate the three pathovars. When tested on non-target DNA, i.e. from plant, bacteria other than X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv. secalis, and culture of microorganisms from wheat grains, the qPCR showed a high specificity. On purified X. translucens pv. undulosa DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification (LAMP) assay. When DNA samples from a set of serial dilutions of X. translucens pv. undulosa cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing ≥1,000 cells. Since 2 µL of the total of 50 µL DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected grain and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one wheat grain. Thus, the qPCR system could detect X. translucens pv. undulosa, X. translucens pv. translucens and/or X. translucens pv. secalis in samples where the bacteria had an average concentration ≥40 cells per grain.

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot is challenging, largely due to the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 × 107 spores/g soil) were stored at various temperatures for two years. Additionally, other samples were buried in soil, or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative polymerase chain reaction (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C, and being buried in the soil were more conductive conditions for spore survival compared to -20°C, 30°C, and at the soil surface. 99.99% of the spores kept on the soil surface were non-viable, suggesting a negative effect of light on spore viability. Additional experiments confirmed the negative effect of UV light on spore viability as spores receiving 2- and 3-hour UV light exhibited lower disease potential and contained less DNA content compared to the untreated control. Finally, this work confirmed that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential due to the presence and persistence of DNA from dead spores.

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Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other brassica crops. Improved understanding of host and pathogen biology is frequently useful in guiding management strategies. In order to better understand infection thresholds, seven-day old seedlings of canola cultivar Westar were inoculated with serially diluted concentrations of P. brassicae resting spores. Controlled soil and plant inoculation assays were performed and the plants maintained in a greenhouse for 42 days and clubroot disease severity evaluated visually. Clubroot symptoms were observed in soils containing as low as one spore/mL soil and on plants inoculated with as few as ≤ 100 resting spores. These thresholds were lower than any previously reported. The results indicated the importance of highly sensitive detection methods for P. brassicae diagnosis and quantification methods for clubroot risk prediction in soils. Furthermore, these results highlighted the low probability of obtaining P. brassicae single spore isolates.

16Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops 17 worldwide. Management of clubroot has been challenging, due largely to the millions of resting 18 spores produced within an infected root that can survive dormant in the soil for many years. This 19 study was conducted to investigate some of the environmental conditions that may affect the 20 survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 × 10 7 21 spores g -1 soil) were stored at various temperatures for two years. Additionally, other samples 22 were buried in soil, or kept on the soil surface in the field. The content of P. brassicae DNA and 23 the numbers of viable spores in the samples were assessed by quantitative polymerase chain 24 reaction (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C 25 and being buried in the soil were better conditions for spore survival than were -20°C, 30°C and 26 at the soil surface. Most of the spores kept on the soil surface were killed, suggesting the 27 negative effect of light on spore viability. Additional experiments confirmed that ultraviolet (UV) 28 light contributed a large negative effect on spore viability as lower pathogenicity and less DNA 29 content were observed from the 2-and 3-hour UV light treated spores compared to the untreated 30 control. Finally, this work demonstrated that DNA-based quantification methods such as qPCR 31 can be poor predictors of P. brassicae disease potential due to the presence and persistence of 32 DNA from dead spores.33 34 Introduction 37 Clubroot, caused by the protist Plasmodiophora brassicae Woronin, is an important threat to 38 Canadian canola (Brassica napus L.) production [1]. In the Canadian Prairies, clubroot was first 39 identified on canola in 2003 in a dozen fields near Edmonton, Alberta [2]. The disease has spread 40 throughout much of the canola producing areas of Alberta [3,4], and has also been confirmed in 41 canola fields in Saskatchewan [5], Manitoba [6], Ontario [7] and North Dakota [8]. 42 43 Many strategies have been proposed for clubroot management and amongst those, crop rotation, 44 along with the use of clubroot-resistant cultivars, was demonstrated to be effective [9]. However, 45 in fields with high resting spore populations, the selection for virulent, but rare, P. brassicae 46 pathotypes can lead to a shift in the population. The once rare virulent pathotype(s) becomes 47 4 59 experience a sharp decline in viability during the first two years without a host [12,20] followed 60 by a slow decline of spore viability over the next 10 to 20 years. Regardless of the kinetics of 61 spore degradation, little is known about which environmental parameters have the greatest 62 effect(s) on spore survival. During the prolonged surviving time, the viability of spores may be 63 impacted by soil type, soil pH values, water content, temperature and light [14]. Other factors 64may include the number of freeze-thaw cycles, rapid temperature shifts, mi...