Abstract
Wheat leaves infected with leaf rust collected across Canada in 2013 were used to isolate 265 Puccinia triticina Eriks. single uredinial isolates. When these were analysed for virulence on 16 standard differential wheat lines 38 virulence phenotypes were found, with MBDS (11.7%), TBBG (11.3%), TNBG (10.2%) and MBTN (8.3%) the most common. In Manitoba and Saskatchewan, 29 virulence phenotypes were found among 236 isolates, with MBDS (13.1%), TBBG (12.7%) and TNBG (11.4%) being the most common. From Ontario, four virulence phenotypes MBTN (73.7%), LCDN (10.5%), MGPS (10.5%) and TCRJ (5.3%) were determined among 19 isolates. There were 10 isolates from Prince Edward Island which grouped into seven different virulence phenotypes, the most common being MBNQ (three isolates) and MCNQ (two isolates). The frequencies of virulence to Lr9, Lr26, Lr3ka, Lr17, Lr30, Lr14a and Lr21 increased, and virulence to Lr2a, Lr2c, Lr16, Lr24, Lr11, Lr10 and Lr18 decreased, when compared with 2012. The increase in virulence frequency to Lr21 is important since many Canadian wheat cultivars have this gene, and could become more susceptible. There were no virulence phenotypes in common between Ontario and Prince Edward Island, and only one virulence phenotype from each of these regions was found in the larger sample from Manitoba and Saskatchewan, demonstrating the differences in the populations across Canada.
Résumé
En 2013, des feuilles de blé infectées par la rouille, collectées partout au Canada, ont été utilisées pour isoler 265 isolats mono-urédiniaux de Puccinia triticina Eriks. Après que ceux-ci ont été analysés quant à leur virulence à l’égard de 16 lignées différentielles de blé, 38 phénotypes de virulence ont été trouvés, MBDS (11.7%), TBBG (11.3%), TNBG (10.2%) et MBTN (8.3%) étant les plus courants. Au Manitoba et en Saskatchewan, 29 phénotypes de virulence ont été trouvés parmi 236 isolats, MBDS (13.1%), TBBG (12.7%) et TNBG (11.4%) étant les plus courants. En Ontario, 4 phénotypes de virulence ont été détectés chez 19 isolats: MBTN (73.7%), LCDN (10.5%), MGPS (10.5%) et TCRJ (5.3%). À l’Île-du-Prince-Édouard, il y avait 10 isolats regroupés en 7 différents phénotypes de virulence, les plus courants étant MBNQ (3 isolats) et MCNQ (2 isolats). Comparativement à 2012, les fréquences de virulence à l’égard des gènes de résistance Lr9, Lr26, Lr3ka, Lr17, Lr30, Lr14a et Lr21 se sont accrues et celles à l’égard des gènes Lr2a, Lr2c, Lr16, Lr24, Lr11, Lr10 et Lr18 ont décru. L’accroissement de la fréquence de la virulence à l’égard du gène Lr21 est importante, puisque plusieurs cultivars de blé canadiens le possèdent et pourraient devenir plus réceptifs. L’Ontario et l’Île-du-Prince-Édouard ne partageaient aucun phénotype de virulence et seulement un phénotype de chacune de ces deux régions a été trouvé dans l’échantillon plus vaste du Manitoba et de la Saskatchewan, ce qui démontre les différences entre les populations de l’ensemble du Canada.
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). Leaf rust severity in Canada changes annually, however susceptible cultivars would sustain damaging yield losses without protection from fungicides. Variation in disease severity is due to several variables including the amount of inoculum in the form of urediniospores blowing into Canada from the USA, temperature, rainfall, relative humidity, genetic resistance of the prevalent cultivars, and foliar fungicide applications.
From field surveys conducted during July to September 2013, wheat leaf rust was found at relatively low levels in Manitoba and Saskatchewan (McCallum and Seto-Goh Citation2014). The crop was seeded early which allowed it to avoid damage from the leaf rust epidemic which developed later in the growing season. In southern Ontario leaf rust was also relatively light, it was found in eight of 40 fields surveyed at a mean severity level of 32% (Xue and Chen Citation2014).
Virulence surveys for P. triticina have been conducted in Canada since 1931. They provide a continuous record of the annual virulence spectrum of the P. triticina population in Canada, which has revealed the evolution of virulence and dynamics of this pathogen population over time (McCallum et al. Citation2016a). The P. triticina population has changed significantly from year to year and evolved new virulence phenotypes continuously over this time period (Wang et al. Citation2010; McCallum et al. Citation2016a). This evolution was characterized by a large number of unique virulence phenotypes each year, with a few predominating for a number of years, then being replaced by other predominant virulence phenotypes. There were also important regional differences with populations from western and eastern Canada differing significantly from each other.
The objective of the present research was to obtain a representative sample of the Canadian P. triticina population in 2013 then determine the frequency of virulence phenotypes and 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, and effectiveness of leaf rust resistance genes (Lr genes) deployed in the Canadian wheat cultivars.
Materials and methods
Virulence on standard seedling differential lines
Infected wheat leaves were collected from individual fields and nurseries from June to September in 2013 in various locations throughout Saskatchewan, Manitoba, Ontario and Prince Edward Island (PEI). 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 urediniospores, water and Tween 20 mixture as described previously (McCallum and Seto-Goh Citation2005). Each pot of ‘Little Club’ seedlings was pre-treated with 50 mL maleic hydrazide solution (0.36 g L−1) ~5 days 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 days 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 days 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-day-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 six 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 pre-treated with maleic hydrazide as described above. 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 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 the differential near-isogenic lines were rated 12 days 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 five ‘Thatcher’ near-isogenic lines (Lr12 [RL6011], Lr13 [RL4031], 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 uredinial isolate, as described previously for the seedling inoculation. Inoculated plants were dried for over 1 h 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 same 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
Virulence on the standard seedling differential lines
From the wheat leaf collections made across Canada in 2013, 265 single uredinial isolates were recovered. When these were tested on the 16 standard seedling differential wheat lines there were 38 unique virulence phenotypes identified. The most common of these were MBDS (11.7%), TBBG (11.3%), TNBG (10.2%) and MBTN (8.3%) (). MBDS was previously one of the most common virulence phenotypes from 2001 to 2004, and it increased in frequency again in 2012 and 2013 (). TBBG was also the most common virulence phenotype in 2012 and was also frequently found in 2004–2005. In contrast TNBG has only been found at high frequencies in recent years ().
Table 1. Frequency and distribution of virulence phenotypes of Puccinia triticina identified in 2013 by infection types to selected resistance genes.
Table 2. Frequency (%) of predominant Puccinia triticina virulence phenotypes in Canada from 2001 to 2013.
From Manitoba and Saskatchewan 29 virulence phenotypes were found among 236 isolates (). The most common of these were MBDS (13.1%), TBBG (12.7%) and TNBG (11.4%), similar to Canada as a whole, since the isolates from this region make up most of the isolates analysed. In 2012 the most common virulence phenotypes were TBBG (22.0%), TNBG (18.6%), TDBJ (10.2%) and MBDS (7.3%) (McCallum et al. Citation2018) so this is similar to 2013 with MBDS becoming more frequent and TBBG, TDBJ and TNBG less frequent. In the neighbouring north-central USA the most common virulence phenotypes in 2013 were TNBGJ (14.2%), MBDSD (11.5%), TBBGJ and TBBGS (both 8.8%) (Kolmer and Hughes Citation2015) which was similar to Manitoba and Saskatchewan.
In Ontario, 19 isolates were analysed and four virulence phenotypes were found, MBTN (73.7%), LCDN (10.5%), MGPS (10.5%) and TCRJ (5.3%) (). In 2012 in Ontario MBTN (34.8%), MCGJ and TDPN (9.1%) were the most common (McCallum et al. Citation2018). MBTN was also common in Ontario in 2011 (25.5%) (McCallum et al. Citation2017) and 2010 (23.7%) (McCallum et al. Citation2016b). While this virulence phenotype was found previously in Quebec and PEI it was not found in Manitoba and Saskatchewan, until 2013 (3.4%). Similarly, in the region of the USA bordering Ontario the most frequently isolated virulence phenotype in 2013 was MBTNB (49.4%) (Kolmer and Hughes Citation2015). Virulence phenotype LCDN was not found in Canada in the previous 20 years, and over that time there were only four other isolates found with the first code letter ‘L’, which are virulent on Lr1, but avirulent on Lr2a, Lr2c and Lr3.
From PEI there were seven virulence phenotypes found among 10 isolates, MBNQ (three isolates), MCNQ (two isolates), MBPS, MCPQ, MCTQ, PCQQ and TBPK (one isolate each) (). The population in PEI has only been sampled in recent years, and only a relatively small number of isolates have been analysed. MBNQ was also found in 2010 (six isolates) and 2008 (one isolate) but was not found in any other region of Canada in recent years. The same is true for MCNQ which was found in PEI in 2009 and 2011 but was not found outside PEI. In 2012 the most common virulence phenotypes from PEI were TBRK (12 isolates) and TBRJ (two isolates), which were not found in 2013. The P. triticina population from PEI appears to be distinct from the populations in Quebec and Ontario with only a few virulence phenotypes in common, and many unique to PEI.
Compared to 2012 there were increases in the frequencies of virulence to Lr9, Lr26, Lr3ka, Lr17, Lr30, Lr14a and a small increase for Lr21 (), while there were decreases to Lr2a, Lr2c, Lr16, Lr24, Lr11, Lr10 and Lr18. The trends over time for virulence to selected resistance genes in the Manitoba and Saskatchewan region are shown in . Virulence to Lr2a was higher in previous years, whereas virulence to Lr9 has been increasing in recent years. Selection in the USA likely accounts for the trend of increasing virulence on Lr9 since this gene is not common in Canadian wheat cultivars. Virulence to Lr24 has been slowly declining since 2007, whereas virulence to Lr17 has been increasing since 2007. There was no virulence to Lr21 prior to 2011, and the frequency of virulence increased in 2012 and 2013, though only slightly. This is significant since many Canadian wheat cultivars have Lr21 such as ‘McKenzie’, ‘Vesper’, ‘Cardale’, ‘AAC Warman’, ‘Snowstar’ and others, these cultivars depend at least partially on its effectiveness to be resistant to leaf rust (McCallum et al. Citation2016a; Toth et al. Citation2018).
Table 3. Frequencies of virulence of Puccinia triticina in Canada in 2013 and 2012 to lines of wheat with single Lr genes for leaf rust resistance.
Fig. 1 Frequency of virulence (%) from 2000–2013a in the Manitoba and Saskatchewan population of Puccinia triticina to near-isogenic lines containing Lr2a, Lr9, Lr16, Lr24, Lr17 or Lr21.
Virulence on adult plant differential lines and additional seedling differential lines
Forty-two isolates, representing all the unique virulence phenotypes found in 2013 were tested on six Thatcher near-isogenic lines at the adult plant stage and 12 additional Thatcher near-isogenic lines at the seedling stage. For the adult plant differentials, all isolates tested were virulent on Lr13, Lr12 (except 169–2 TBPK) and Lr37 (except 94–1 PBJQ, 173–2 TBBG, 169–2 TBPK, 105–1 TDGJ), whereas all isolates were avirulent on Lr22a. All isolates were avirulent to Lr35 (except 169–1 MBPS, 128–1 MBRJ, 108–1 MBTN). This is similar to previous years with high frequencies of virulence to Lr13, Lr12 and Lr37, a low frequency of virulence to Lr35, and no virulence to Lr22a. The resistance gene Lr22a is present in some Canadian cultivars such as ‘AC Minto’, ‘5500HR’ and ‘5600HR’ (Hiebert et al. Citation2007), although it was not found commonly among more modern cultivars (Toth et al. Citation2018).
For the additional seedling differentials all isolates tested were virulent to Lr3bg (except 128–1 MBRJ, 116–1 TBBJ, 3–3 TBBJ, 169–2 TBPJ, 3–1 TBBJ, 46–1 TNBG), Lr14b (except 94–1 PBJQ), Lr15 and Lr20 (except 58–1 TBBG, 92–2 TDBG, 57–1 TLBG, 56–2 TNBG), whereas all isolates were avirulent to Lr19, Lr23 (except 47–1 MLDS), Lr25 (except 94–1 PBJQ), Lr28 (except 128–1 MBRJ, 94–1 PBJQ, 58–1 TBBG, 3–3 TBBJ, 169–2 TBPK, 92–2 TDBG, 50–1 TDBJ, 52–1 TDBJ, 105–1 TDGJ, 3–1 TDBJ, 57–1 TLBG, 46–1 TNBG, 56–2 TNBG, 48–2 TNBJ), Lr29, Lr32 and Lr52. All isolates reacted similarly to Lr2b as they did to Lr2a except 94–1 PBJQ which was avirulent to Lr2a but virulent to Lr2b. These results are also similar to previous years with very low levels or no virulence to Lr19, Lr23, Lr25, Lr28, Lr29, Lr32 and Lr52.
The P. triticina population in Canada in 2013 changed from that found in 2012 and previous years. MBDS again became the most common virulence phenotype, after being the most common virulence phenotype in 2002 then declining to low levels from 2005 to 2011 (). TBBG, TDBJ and TNBG were all common in 2013, as they were in 2012. The samples from Ontario and PEI differed from each other, but both also differed from those found in western Canada. Of the four virulence phenotypes found in Ontario all were unique to Ontario, except MBTN, and all the virulence phenotypes found in PEI were unique to PEI, except MBPS. The frequencies of virulence to most resistance genes followed trends from recent years. The most significant finding is the increase in virulence of Lr21, which could render the cultivars carrying this gene more susceptible to leaf rust.
The P. triticina population in Canada originates each year from the USA as urediniospores are blown northward into Canada from wheat growing regions in the neighbouring states. Therefore the population found in Manitoba and Saskatchewan is normally similar to that found in North and South Dakota and the populations in Ontario and Quebec are similar to those in the north-eastern USA. Additionally there may be some selection for virulent phenotypes within Canada. The common resistance genes in Canadian wheat cultivars include Lr1, Lr2a, Lr10, Lr13, Lr14a, Lr16, Lr21 and Lr34 (McCallum et al. Citation2016a; Toth et al. Citation2018), virulence is normally at a high level for these genes as in 2013, except for Lr16, Lr21 and Lr34. While nearly all isolates are classified as avirulent to Lr16 they typically have an intermediate infection type ‘1+’ and the Thatcher-Lr16 line suffers significant infection in the field of ~60% flag leaf infection compared with Thatcher at 80%. Selection likely occurs on wheat cultivars in Canada with Lr21, however many cultivars grown in the north-central USA also have Lr21 which has an influence on the population entering Canada.
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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