Phytoplasma dynamics in Aster Yellows-infested Brassica plants as determined by droplet digital PCR (ddPCR). M. H. BAHAR, C. OLIVIER, D. BEKKAOUI, D. HEGEDUS AND J. SOROKA. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
The incremental incidence of Aster Yellows (AY) disease caused by phytoplasma (a non-cellular bacteria-like organism) in North America is of great concern. Although there is no prescribed control measure for AY, monitoring and phytosanitary activities can prevent its spread. Knowledge of phytoplasma distribution across plant parts is important to optimize sampling for diagnosis required by phytosanitary regulations, but such information is not available for AY-diseased Brassica plants. The numbers of phytoplasmas present in different locations (root, stem above 10 cm of soil, stem above 35 cm of soil, older leaf, younger leaf, petiole, flower and pod) of field-collected naturally AY-diseased Brassica plants (B. napus L., B. alba, B. carinata, Camelina sativa and Thlaspi arvense), and of artificially inoculated laboratory-grown canola (B. napus) plants over the growing period were quantified by droplet digital PCR (ddPCR). Phytoplasmas were detected in every part of infested plants. The numbers of phytoplasmas varied among parts of both field-collected and artificially inoculated canola plants, with more phytoplasmas in the lower parts (root) of younger plants and in the reproductive parts (pod) of older plants. The severity of AY disease symptoms during bolting, flowering and seed-set related directly to the numbers of phytoplasmas in the plant tissues. This result will be useful for early season monitoring of AY in canola fields.
In vivo imaging of Plasmodiophora brassicae infection in Arabidopsis using a fluorescence probe. J. D. BUSH, Z. ZHOU AND Y. WEI. Department of Biology, W.P. Thompson Building, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada
Clubroot is one of the most destructive plant diseases in modern agriculture and poses a significant threat to prairie canola production. Despite many attempts to understand the biology of the causative agent, Plasmodiophora brassicae Woronin, infection strategies unique to the pathogen continue to elude researchers. The most discerning obstacle in P. brassicae research is the inability to perform in vivo imaging of the pathogen during the infection process. Here, we report the first technique for in vivo imaging of P. brassicae in infected root tissues using a pathogen-specific stain. Transmission electron microscopy of Arabidopsis host tissues reveals that the pathogen possesses unique subcellular lipid compartmentalization during all developmental stages. We confirmed this through a histological examination using Sudan Black 3 staining. We hypothesize the lipid body structures are sourced from the pathogen as host tissues including root hairs and epidermal cells rarely contain these structures. Fluorescence microscopy of infected and uninfected hosts using several lipid dyes indicates Nile red (9-diethylamino-5[H]-benzo(α)phenoazine) as a fluorescence probe suitable for live cell imaging of the pathosystem. This technique will allow for visual examination of the pathogen’s biology and for understanding the cellular level interactions between P. brassicae and host plants, especially when using fluorescence protein-tagged Arabidopsis lines.
Length polymorphisms in the IGS region distinguishes two pathogenic races of Colletotrichum truncatum from lentil. J. M. H. DURKIN, J. BISSETT, R. SONG, H. PAHLAVANI, B. MOONEY AND L. BUCHWALDT. Saskatoon Research Centre, Agriculture and Agri-Food Canada (AAFC), 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (J.B.) Eastern Cereal and Oilseed Research Centre, AAFC, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada; and (H.P.) Gorgan University of Agricultural Sciences and Natural Resources, Golestan, PO 386, Iran
Colletotrichum truncatum (Schwein.) Andrus & Moore causes anthracnose of lentil (Lens culinaris subsp. culinaris Medic). Two races, Ct0 and Ct1, have been identified in Canada by inoculation of differential lentil lines. Our objective was to develop a PCR-based test to differentiate these races. Since polymorphisms in the Internal Transcribed Spacer (ITS) and Intergenic Spacer Region (IGS) in ribosomal DNA are used to differentiate many fungal species, we initially used universal primers to amplify these regions from race Ct0 and Ct1 isolates. DNA sequencing and alignment of the amplicons showed a polymorphic region in IGS consisting of 2–12 repeats of a 39 nucleotide (nt) minisatellite. Subsequently, a new primer pair was designed around this length polymorphism using forward primer (39F) GAGATAAGTAAAGACG GAGATAAA and reverse primer (39R) TAGGCGCCAAGGTAGAAAGT. This probe amplified a varying number of 2, 4, 5, 6, 8 or 12 repeats of the minisatellite in a new set of Ct0 isolates using gel electrophoresis, while Ct1 isolates showed a single band of either 7 or 9 repeats. Each 39 nt repeat forms a stable hairpin structure, which explains the lack of other polymorphisms in this region. A survey of 208 isolates collected from seed in 74 lentil fields showed that race Ct0 constituted 95% of the population in 2012, whereas the two races were equally common in 2004. Two published primers presumed to be specific to C. truncatum from lentil did not amplify the expected DNA sequence in 19% and 30% of our isolates.
Leaf spotting for common and durum wheat in Saskatchewan – effect of soil zone and previous crop. S. LIM, M. R. FERNANDEZ, F. L. DOKKEN-BOUCHARD, S. G. MILLER, K. HODGE AND C. PELUOLA. Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food Canada (AAFC), P.O. Box 1030, Swift Current, SK S9H 3X2, Canada; (F.L.D.-B., S.G.M.) Crops and Irrigation Branch, Saskatchewan Ministry of Agriculture, 3085 Albert Street, Regina, SK S4S 0B1, Canada; (K.H.) Knowledge Technology Transfer Office, AAFC, #300-2010 12th Avenue, Regina, SK S4P 0M3, Canada; and (C.P.) Crop Protection Laboratory, Crops and Irrigation Branch, Saskatchewan Ministry of Agriculture, 346 McDonald Street, Regina, SK S4N 6P6, Canada
The leaf spotting complex (LS) is the most frequently encountered wheat disease across the Prairies. A 12-year survey (2001–2012) was conducted in all 20 crop districts of Saskatchewan to determine differences for LS severity and causal pathogens between common and durum wheat for different soil zones and previous crops. Percentage flag leaf area affected by LS was determined in 1320 common and 349 durum wheat fields. Each year, fungi were identified and quantified in the most affected fields. Pyrenophora tritici-repentis (Died.) Drechs. (Ptr) was most commonly isolated across all soil zones, followed by Phaeosphaeria nodorum (Müll.) Hedjar. (Pn), Mycosphaerella graminicola (Fuckel) Schröt. in Cohn (Mg), Cochliobolus sativus (Ito & Kurib.) Drechs. ex Dastur (Cs), and Phaeosphaeria avenaria (Weber) Erikss. (Pa). For the Dark Brown soil zone, greater isolations of Pn and Mg occurred on common than durum wheat, while greater isolations of Cs and Pa occurred on durum wheat. For common wheat, LS severity was lowest in the Brown soil zone (BRSZ) and highest in the Black soil zone (BLSZ) where Ptr isolation was lowest, while Mg was higher in the BLSZ than the BRSZ, and Cs isolation was lowest in the BRSZ. There were inconsistencies in previous crop responses; however, multivariate analysis showed that for common wheat, previous oilseed crops were associated more with Pn than Ptr in all soil zones. This long-term survey showed that the effect of wheat species and previous crop on LS severity and pathogen isolation depended on the soil zone.
Stripe rust management of wheat – fungicide application timing, cultivar resistance and effect of seeding date. J. LIU, J. TAYLOR, T. DAMENT, J. T. VERA AND H. R. KUTCHER. Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada
Stripe rust [Puccinnia striiformis Westend. f. sp. tritici Eriks.] of wheat is now observed each season in Saskatchewan. Our objectives were to determine: the benefit of fungicide on wheat cultivars varying in resistance to stripe rust; the appropriate fungicide timing; and the effect of seeding date on disease severity, yield and quality. The study was conducted in replicated field experiments at Saskatoon and Pike Lake, SK using the cultivars ‘AC Barrie’ (susceptible), ‘CDC Image’ (moderately resistant) and ‘Lillian’ (resistant), seeded early (mid-May) or late (early June, 2013). Tebuconazole (Folicur 250EW) was applied at 0.5 L ha−1 at stem elongation, flowering (50% of heads in anthesis), early milk or all three stages. Fungicide reduced disease severity and increased yield and quality of ‘AC Barrie’; the trend was similar for ‘CDC Imagine’, but few symptoms occurred on treated or non-treated ‘Lillian’ and no yield or quality benefit was observed for this cultivar. The most appropriate fungicide application time was the flowering stage of wheat, and three applications did not improve disease control, yield or quality over this single application. Early seeding, compared with late, had less severe stripe rust and greater yield (56% greater at Saskatoon and 34% at Pike Lake). Yield of ‘Lillian’ and ‘CDC Imagine’ were 16% greater than ‘AC Barrie’ at Saskatoon, but the difference was less than 3% at Pike Lake. Seeding in May and applying fungicide at flowering increased yield of ‘AC Barrie’ by 38%; however, fungicide did not increase yield of ‘CDC Imagine’ and ‘Lillian’.
Blackleg of canola – new management strategies against an old disease in western Canada. G. PENG, W. G. D. FERNANDO, R. LANGE AND H. R. KUTCHER. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; (W.G.D.F.) Department of Plant Sciences, University of Manitoba, 222 Agriculture Building, Winnipeg, MB R3T 2N2, Canada; (R.L.) Alberta Innovates, P.O. Bag 4000, Vegreville, AB T9C 1T4, Canada; and (H.R.K.) Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada.
Blackleg [Leptosphaeria maculans (Desmaz.) Ces. & de Not.] is a threat to canola (Brassica napus L.) production in western Canada. For many years, this disease was successfully managed with resistant cultivars and >3-year crop rotations. Over the past few years, severe blackleg disease has been reported on R- or MR-rated canola cultivars. To better understand the disease dynamics and mitigate the risk of widespread resistance breakdown, several studies were carried out to identify specific R genes in Canadian canola lines, assess the frequency and distribution of L. maculans avirulence (Avr) alleles in the pathogen population, and evaluate fungicides and application timing for blackleg management. A very limited number of R genes were identified, with Rlm3 and Rlm1 being found in 65% and 10% of the lines evaluated. Other R genes were infrequent. In the pathogen population, very few isolates (<5%) carried the AvrLm3 or AvrLm1 allele in most regions, and this indicates that the specific R genes Rlm3 and Rlm1 are ineffective. The Avr profile is being analysed for L. maculans on R-rated cultivars suffering severe blackleg to better understand the dynamic of pathogen population and cultivar resistance in commercial fields. Early application of a fungicide at the 2–4 leaf stage reduced blackleg severity with inconsistent yield benefit, depending on the cultivar resistance or disease level. These studies, once completed, will improve our understanding of the current pathogen population, cultivar resistance characteristics, and risk mitigation options for management of blackleg disease.
Transcriptome analysis of the clubroot-resistance gene Rpb1 using high-throughput RNA sequencing. T. SONG, R. LAHLALI, F. YU, B. D. GOSSEN AND G. PENG. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada
Clubroot [Plasmodiophora brassicae Woronin] disease is a serious threat to canola (Brassica napus L.) production in western Canada. Use of clubroot resistant cultivars is the most effective and economical strategy to minimize the impact of this disease. Several clubroot resistance (CR) loci have been identified from various Brassica species, but few reports examined the molecular mechanisms behind these CR genes. In this study, we conducted a transcriptome analysis of the CR gene Rpb1 originating from pak choy (B. rapa ssp. chinensis) cv. ‘Flower Nabana’ using high-throughput RNA sequencing (RNA-seq). Transcriptome profiling revealed that Rpb1-mediated resistance to clubroot was composed of both pathogen-induced defence responses and suppression of host metabolism that favoured pathogenesis. Several host defence-related biological processes were stimulated in the resistant cultivar upon pathogen infection, including defensive callose deposition, enhanced signalling and metabolism of the phytohormones jasmonate and ethylene, and secondary metabolism possibly generating anti-microbial compounds. In contrast, several pathogenesis-promoting metabolic pathways were suppressed in resistant plants relative to susceptible plants, including the auxin signalling pathways and regulation of cell growth. Also, several differentially expressed genes were characterized as defence-related transcription factors, possibly regulating the altered metabolisms/pathways in resistant plants. The RNA-seq analysis provides important insights into the molecular mechanisms of clubroot resistance in Brassica.