Abstract
Wheat leaves infected with Puccinia triticina, the causal agent of wheat leaf rust, were collected from across Canada in 2014. From these leaves, 102 single-pustule isolates were recovered and tested for virulence on 16 standard differential wheat lines, with 28 unique virulence phenotypes found. The most common were TBDG (16.7%), TBBG (13.7%), MLPS and TNBG (both at 7.8%). Most isolates (95) originated from Manitoba and Saskatchewan, and the most common phenotypes found in this region were TBDG (17.9%), TBBG (14.7%), MLPS and TNBG (both at 8.4%). From Ontario, six virulence phenotypes were found from seven isolates: TCGJ (two isolates), MBDS, MCQG, MFPS, MGBS and TBGS (one isolate each). Frequencies of virulence in 2014 increased for Lr2a, Lr2c, Lr26 and Lr10 when compared with 2013, while there were decreases in virulence to Lr9, Lr24, Lr3ka, Lr30, Lr14a, Lr21 and LrCen. When 30 isolates, representing most of the unique virulence phenotypes, were tested on adult plants nearly all were virulent to the adult plant resistance genes Lr12, Lr13 and Lr37, most were avirulent to Lr35, and all were avirulent to Lr22a. When this subset of isolates was inoculated onto additional seedling differentials, most were virulent to Lr15 and Lr14b and avirulent to Lr25 and Lr29, and all isolates were avirulent to Lr19, Lr32 and Lr52. There were intermediate levels of virulence on Lr3bg, Lr20, Lr23 and Lr28.
Résumé
Des feuilles de blé infectées par Puccinia triticina, l’agent causal de la rouille brune, ont été collectées en 2014 partout au Canada. De ces feuilles, 102 isolats à pustule unique ont été récupérés et testés pour leur virulence à l’égard de 16 lignées différentielles de blé, à la suite de quoi 28 phénotypes uniques de virulence ont été trouvés. Les plus courants étaient TBDG (16.7%), TBBG (13.7%), MLPS et TNBG (7.8% chacun). La plupart des isolats (95) provenaient du Manitoba et de la Saskatchewan et les phénotypes les plus courants trouvés dans cette région étaient TBDG (17.9%), TBBG (14.7%), MLPS et TNBG (8.4% chacun). En Ontario, on a trouvé six phénotypes de virulence chez sept isolats: TCGJ (deux isolats), MBDS, MCQG, MFPS, MGBS et TBGS (un isolat chacun). En 2014, les fréquences de virulence se sont accrues à l’égard des gènes Lr2a, Lr2c, Lr26 et Lr10, comparativement à 2013, tandis qu’elles ont baissé à l’égard des gènes Lr9, Lr24, Lr3ka, Lr30, Lr14a, Lr21 et LrCen. Lorsque 30 isolats, représentant la majorité des phénotypes uniques de virulence, ont été testés sur des plants adultes, ils étaient presque tous virulents à l’égard des gènes de résistance Lr12, Lr13 et Lr37 de ces plants, la plupart n’étaient pas virulents à l’égard du gène Lr35, et aucun n’était virulent à l’égard du gène Lr22a. Lorsque ce sous-ensemble d’isolats a été testé sur des lignées différentielles supplémentaires au stade de semis, la plupart étaient virulents à l’égard des gènes Lr15 et Lr14b et non virulents à l’égard des gènes Lr25 et Lr29, et aucun des isolats n’était virulent à l’égard des gènes Lr19, Lr32 et Lr52. Par ailleurs, il y avait des degrés intermédiaires de virulence à l’égard des gènes Lr3bg, Lr20, Lr23 et Lr28.
Introduction
Wheat leaf rust, caused by Puccinia triticina Eriks. (Anikster et al. Citation1997) (syn. P. recondita Rob. ex Desmaz. f. sp. tritici), is an annual production concern for wheat growers in Canada, and is one of the most common and damaging diseases of wheat worldwide (Huerta-Espino et al. Citation2011). The severity of the epidemic in Canada varies annually, but the disease is normally found widespread throughout eastern Saskatchewan, Manitoba, Ontario, Quebec and the Maritime provinces. Variables affecting the severity of the epidemic include the amount of inoculum in the form of urediniospores entering Canada, temperature, rainfall and the relative susceptibility of the wheat crop.
Annual virulence surveys for P. triticina done in Canada since 1931 have generated a continuous record of the annual virulence spectrum of the P. triticina population, which revealed the evolution of virulence and dynamics in this population over time (McCallum et al. Citation2016a). This population changed significantly from year to year and continuously diversified over this time period (Wang et al. Citation2010; McCallum et al. Citation2016a). There are typically over 30 unique virulence phenotypes found annually, with a few predominating for a few years, then being replaced by other predominant virulence phenotypes. The populations in western Canada differ significantly from those found in eastern Canada, and the population found in Prince Edward Island also appears to be unique (McCallum et al. Citation2018, Citation2019).
The objective of the current study was to analyse the virulence spectrum from a representative sample of the Canadian P. triticina population in 2014, and determine the frequency of virulence to key resistance genes. Results were compared between regions and with previous years from the same region to detect trends in virulence changes and the predominant virulence phenotypes.
Materials and methods
Virulence on the standard seedling differential lines
Infected wheat leaves were collected from individual fields and nurseries from June to September in 2014 in various locations throughout Saskatchewan, Manitoba and Ontario. The leaves were air dried at 20–27°C for ~12–24 h then stored separately for 2–4 months at 5°C. Puccinia triticina urediniospores from individual collections were then scraped off from infected leaves using a metal spatula and a small amount of water, and inoculated onto the susceptible wheat cultivar ‘Little Club’ by rubbing the leaves with a urediniospores, water and Tween 20 mixture as described previously (McCallum and Seto-Goh Citation2005). Each pot of ‘Little Club’ seedlings was pretreated with 50 mL maleic hydrazide solution (0.36 g L−1 concentration) ~5 d after seeding to prevent the emergence of secondary leaves and to produce larger uredinia with abundant sporulation. A plastic cone ~25 cm in height with an open top was placed over each pot to minimize cross-contamination. Inoculated plants were placed into a dew chamber (Model I-60D, Percival Scientific, Perry, IA) with nearly 100% relative humidity for ~17 h to allow the urediniospores to germinate and initiate the infection process, then placed into a greenhouse at 20 ± 4°C with supplemental high-pressure sodium lighting, resulting in a photoperiod of ~16 h. Approximately 7 d after inoculation, chlorotic spots appeared, indicating areas of infection. The leaves were then trimmed so that a single isolated uredinium remained on the upper edge of each trimmed leaf. Cross-contamination was minimized by removing all extra leaves.
At ~14 d after inoculation, urediniospores were collected from a single isolated uredinium into a 00 gelatin capsule using a vacuum suction micro-collector, mixed with a light mineral oil (Bayol, Esso Canada, Toronto, ON), and sprayed onto a 7-d-old set of wheat seedlings which included a flat of ‘Thatcher’ and 16 single resistance gene ‘Thatcher’ near-isogenic lines to test virulence, and a pot of ‘Thatcher’ plants for urediniospore increase. Two single uredinial isolates were typically evaluated from each rust collection, although sometimes one or three isolates per collection were analysed. The inoculated pot of ‘Thatcher’ increase was kept isolated with a plastic cone on top of the pot, and urediniospores were vacuum collected for subsequent inoculations. Approximately 12 seeds of each ‘Thatcher’ near-isogenic line were planted in a clump, and the clumps were evenly spaced in a fibre flat (25 × 15 cm). Plants were pretreated with maleic hydrazide as described above. This treatment resulted in larger pustules but did not change the infection types. After inoculation, the plants were allowed to dry for at least 1 h to allow the oil to evaporate and then incubated and maintained as described above. Infection types produced on the 12 standard leaf rust (Lr) differential lines (Set 1: Lr1 (RL6003a), Lr2a (RL6016), Lr2c (RL6047), Lr3 (RL6002); Set 2: Lr9 (RL6010), Lr16 (RL6005), Lr24 (RL6064), Lr26 (RL6078); Set 3: Lr3ka (RL6007), Lr11 (RL6053), Lr17 (RL6008), Lr30 (RL6049)) were used to determine the three letter code according to the virulence phenotype nomenclature (Long and Kolmer Citation1989). Four supplemental differential lines (Set 4: LrB (RL6051), Lr10 (RL6004), Lr14a (RL6013), Lr18 (RL6009)) were added to provide additional virulence information about the isolates, resulting in a four letter code. All isolates were also inoculated onto ‘Thatcher’, Thatcher-Lr21 (RL6043) and Thatcher-LrCen (RL6003b). The resistance gene temporarily named LrCen was previously identified in the Thatcher-Lr1 near-isogenic line RL6003 (McCallum and Seto-Goh Citation2006b). Infection types on all differential near-isogenic lines were rated 12 d after inoculation. Isolates that produced infection types ‘;’ (hypersensitive flecks), ‘1’ (small uredinia with necrosis) and ‘2’ (small- to medium-sized uredinia with chlorosis) were considered avirulent to the differential line, and those that produced infection types ‘3’ (medium-sized uredinia without chlorosis or necrosis) and ‘4’ (large uredinia without chlorosis or necrosis) were considered virulent to the line (Long and Kolmer Citation1989). Inoculations were repeated if the infection response was not clear.
Virulence on adult plant differential lines and additional seedling differential lines
At least one isolate from most of the unique virulence phenotypes identified was inoculated onto adult plants of ‘Thatcher’ and six ‘Thatcher’ near-isogenic lines (Lr12 [RL6011], Lr13 [RL4031], Lr21 [RL6043], Lr22a [RL6044], Lr35 [RL6082] or Lr37 [RL6081]), using urediniospores that were increased as described previously. Single plants of each ‘Thatcher’ near-isogenic line and ‘Thatcher’ were grown together in a 15-cm-diameter pot in a greenhouse at day/night temperatures of 25/18°C with supplemental high-pressure sodium lighting. Plants were trimmed so that only two or three culms per plant remained. Flag leaves of all the plants within a pot were inoculated with a single pustule isolate, as described previously for the seedling inoculation. Inoculated plants were dried for over one hour to prevent cross-contamination and then incubated overnight in a dew chamber and grown in the greenhouse as described previously for seedling inoculation. Infection types were evaluated 14 d after inoculation. This subset of isolates was also tested on 12 additional ‘Thatcher’ near-isogenic lines at the seedling stage (Lr2b [RL6019], Lr3bg [RL6042], Lr14b [RL6006], Lr15 [RL6052], Lr19 [RL6040], Lr20 [RL6092], Lr23 [RL6012], Lr25 [RL6084], Lr28 [RL6079], Lr29 [RL6080], Lr32 [RL6086] and Lr52) and retested on Lr21 (RL6043). These isolates were also retested on seedling plants of the set of 16 ‘Thatcher’ near-isogenic lines mentioned previously to confirm their infection types, since many of these resistance genes (particularly Lr18 and LrB) are sensitive to temperature and other conditions. Inoculation, incubation and rating were as described previously for seedling evaluation.
Results and discussion
Wheat leaf rust was found at moderate to high levels in field surveys in Manitoba and eastern Saskatchewan in June to September 2014 with an average severity level of 30% of the flag leaf infected in Manitoba and 15% in Saskatchewan (on susceptible cultivars which had not been treated with fungicide), though the epidemic developed later in the growing season than usual (McCallum and Reimer Citation2015). In Ontario leaf rust was found in 12 of 31 fields surveyed with a mean severity level of 3.5% (Xue and Chen Citation2015).
Virulence on the standard seedling differential lines
There were 28 different virulence phenotypes found from 102 single pustule isolates made from leaf rust infected wheat samples collected across Canada in 2014 (). The most commonly found were TBDG (16.7%), TBBG (13.7%), MLPS and TNBG (both at 7.8%). TBDG was rarely found in previous years, whereas TBBG was common from 2004 to 2005 and from 2010 to 2014 (). MLPS was not found at high frequencies in recent years, but TNBG was one of the most common virulence phenotypes found in 2012 and 2013. All the virulence phenotypes found in Canada during 2014 were previously found over the years 1995 to 2013 with the exceptions of TNBH and TNJQ. These appear to have one or more virulence changes from common phenotypes such as TNBG and TNBJ.
Table 1. Frequency and distribution of virulence phenotypes of Puccinia triticina identified in 2014 by infection types to selected resistance genes
Table 2. Frequency (%) of predominant Puccinia triticina virulence phenotypes in Canada from 2001 to 2014
Most of the isolates analysed in 2014 (95) were from Manitoba and Saskatchewan. There were 24 different virulence phenotypes found among these 95 single pustule isolates (). The most common virulence phenotypes from this region were TBDG (17.9%), TBBG (14.7%) and MLPS and TNBG (both at 8.4%). In 2013 the most common virulence phenotypes from this region were MBDS (13.1%), TBBG (12.7%) and TNBG (11.4%). MBDS was also common in 2014 (6.3%) and has been one of the most common virulence phenotypes in Canada from 2012 to 2014 and from 2001 to 2004 (). In the region bordering the USA, the most frequent virulence phenotypes in 2014 were TBBGS (36.5%), MBTNB (13.9%) and TNBJJ (7.0%) (Kolmer and Hughes Citation2016). Of these, TBBG (14.7%) and MBTN (6.3%) were also common in Manitoba and Saskatchewan, while TNBJ was less frequently found (2.1%). Inoculum enters Manitoba and Saskatchewan from neighbouring USA states and the virulence phenotypes found are often similar.
From the seven isolates analysed from Ontario there were six virulence phenotypes identified: TCGJ (two isolates), MBDS, MCQG, MFPS, MGBS and TBGS (one isolate each) (). While this is a relatively small sample, four of these virulence phenotypes (TCGJ, MCQG, MGBS and TBGS) were not found in Manitoba and Saskatchewan. In 2013 the most common virulence phenotypes found in Ontario were MBTN (73.7%), LCDN and MGPS (both 10.5%) though there were only 19 isolates analysed in 2013 (McCallum et al. Citation2019). In the neighbouring USA states that border Ontario the most common virulence phenotypes in 2014 were MBTN (53.1%), MCTNB (18.8%) and TNBGJ (12.5%) (Kolmer and Hughes Citation2016). None of these were found in Ontario in 2014, though the sample size of seven isolates was small.
There were increases in the frequencies of virulence to Lr2a, Lr2c, Lr26 and Lr10 when comparing 2014 with 2013, while there were decreases in virulence to Lr9, Lr24, Lr3ka, Lr30, Lr14a, Lr21 and LrCen (). The increases in virulence to Lr2a and Lr2c indicate that T _ _ _ virulence phenotypes became more frequent relative to M _ _ _ virulence phenotypes. The decrease in the frequency of Lr24 virulence in 2014 follows a trend that started in 2008 in Manitoba and Saskatchewan (). The decline in virulence frequency to Lr21 in 2014 is a bit surprising since it had been increasing annually in Manitoba and Saskatchewan since 2010 (). This is also important to the Canadian wheat industry since many cultivars grown in Canada have Lr21, such as ‘McKenzie’, ‘Vesper’, ‘Cardale’, ‘AAC Warman’ and others (McCallum et al. Citation2016a; Toth et al. Citation2018). An increasing frequency of virulence to Lr21 observed from 2011–2013 could make these cultivars more susceptible, although this virulence frequency declined in 2014.
Table 3. Frequencies of virulence of Puccinia triticina in Canada in 2014 and 2013 to lines of wheat with single Lr genes for leaf rust resistance
Fig. 1 Frequency of virulence (%) from 2000–2014 in the Manitoba and Saskatchewan population of P. triticina to near-isogenic lines containing Lr2a, Lr9, Lr16, Lr24, Lr17 or Lr21. Data from McCallum and Seto-Goh (Citation2003, Citation2004, Citation2005, Citation2006a, Citation2006b, Citation2008, Citation2009) and McCallum et al. (Citation2010, Citation2011, Citation2013, Citation2016b, Citation2017, Citation2018, Citation2019)
Virulence on adult plant differential lines and additional seedling differential lines
Thirty isolates representing most of the unique virulence phenotypes found from testing with the standard seedling differential set were inoculated onto six Thatcher differential lines with adult plant resistance genes at the adult plant stage, and onto 12 additional seedling differentials at the seedling stage. Most isolates were virulent to the adult plant resistance genes Lr12 (except 26-1 LGBS, 236-1 TCGJ, 276-3 MGBS), Lr13 (except 46-1 TDBJ, 205-2 MDTS and 276-1 TBGS), and Lr37 (except 26-1 LGBS, 46-1 TDBJ, 166-1 TDBJ, 229-1 PBDG, 230-1 PCDQ, 230-2 PCDQ, 236-1 TCGJ and 276-3 MGBS). All isolates were avirulent to Lr22a and Lr35 (except 26-1 LGBS). When inoculated onto additional seedling differentials all isolates were virulent to Lr15 and Lr14b (except 178-1 PBDG, 229-1 PBDG, 230-1 PCDQ and 230-2 PCDQ), and avirulent to Lr19, Lr25 (except 178-1 PBDG, 229-1 PBDG, 230-1 PCDQ), Lr29 (except 122-1 TBBG), Lr32 and Lr52. All isolates had a similar infection type on Lr2b as they did for Lr2a except for 178-1 PBDG, which was virulent on Lr2b but avirulent on Lr2a. There were intermediate levels of virulence on Lr3bg, Lr20, Lr23 and Lr28 with 20, 24, 13 and 19 isolates virulent out of the 30 tested, respectively on each of these lines.
Overall the P. triticina population found in Canada during 2014 was similar to 2013. TBDG was the most common virulence phenotype, though it was rarely found previously, whereas phenotypes TBBG and TNBG were found frequently in recent years. The relatively small sample from Ontario was diverse and different from western Canada, similar to previous years. There were changes in the frequencies of virulence for many of the resistance genes, but decreases in the frequencies of virulence to Lr2a, Lr2b and Lr21 were the most significant. Changes to the level of virulence on the common resistance genes in Canadian wheat cultivars (Lr1, Lr2a, Lr10, Lr13, Lr14a, Lr16, Lr21 and Lr34) can result in increased susceptibility in these cultivars. For example, the increase in Lr21 virulence has resulted in increased susceptibility on Lr21 carrying cultivars such as ‘McKenzie’ and ‘Glenn’. Most resistance genes were ineffective against a portion of the Canadian P. triticina population except for Lr19, Lr22a, Lr32 and Lr52, which were resistant to all isolates to which they were tested.
Acknowledgements
We thank André Comeau, Harpinder Randhawa and Richard Martin who sent samples for analysis, and other cooperators who grew trap rust nurseries.
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