Abstract
Leaf rust collections were made across Canada in 2011 and 287 single-pustule isolates were tested for virulence on 16 standard differential lines and two additional lines containing Lr21 and LrCen, respectively. Of the 70 different virulence phenotypes found in Canada during 2011, the most common were TDBJ (12.5%), TDBG (10.5%) and MLDS (7.7%), which is similar to the findings from 2010. Three isolates from Alberta each had a unique virulence phenotype. From Manitoba and Saskatchewan, 33 virulence phenotypes were found among 216 isolates, with the most common being TDBJ (16.7%), TDBG (13.9%), MLDS and TBBG (both at 9.3%). There were 29 virulence phenotypes among 47 isolates from Ontario, with MBTN (25.5%), MCTN (8.5%) predominating. Ten virulence phenotypes were found from 11 isolates in Quebec, and six virulence phenotypes among 10 isolates from Prince Edward Island. Compared with 2010, there were increases in the frequencies of virulent isolates in Canada to Lr2a, Lr2c, Lr16, Lr26, Lr3ka, Lr11 and Lr30 while there were declines in the frequencies of virulent isolates to Lr9, Lr24, Lr10 and Lr14a. When a group of 161 representative isolates were tested on five adult plant differentials, all isolates were avirulent to Lr22a, most were virulent to Lr12, Lr13 and Lr37, while only 16 were virulent to Lr35. When this same group of isolates was tested on 12 additional lines at the seedling stage, all isolates were avirulent to Lr19, Lr32, Lr29 and Lr52 and virulent to Lr15, while they differed in their reactions to Lr2b, Lr3bg, Lr14b, Lr20, Lr23, Lr25 and Lr28. Virulence for Lr21 was detected for the first time in Canada from five different virulence phenotypes, though at a low frequency (3.8%). This finding has implications for wheat breeding in Canada, since Lr21 had been completely effective since its release in the cultivar ‘AC Cora’ in 1994.
Résumé
En 2011, des échantillons de rouille brune ont été collectés partout au Canada et 287 isolats obtenus d’une pustule unique ont été testés pour la virulence sur 16 lignées différentielles standards et 2 lignées additionnelles comprenant les gènes de résistance Lr21 et LrCen, respectivement. Des 70 différents phénotypes de virulence trouvés au Canada en 2011, les plus courants étaient TDBJ (12.5%), TDBG (10.5%) et MLDS (7.7%), ce qui ressemble aux résultats de 2010. Trois isolats provenant d’Alberta possédaient chacun un phénotype de virulence unique. Parmi les 216 isolats provenant du Manitoba et de la Saskatchewan, 33 phénotypes de virulence ont été détectés, TDBJ (16.7%), TDBG (13.9%), MLDS et TBBG (9.3% chacun) étant les plus courants. Il y avait 29 phénotypes de virulence parmi les 47 isolats provenant d’Ontario, MBTN (25.5%) et MCTN (8.5%) prédominant. Du Québec, on a trouvé 10 phénotypes de virulence parmi les 11 isolats et, de l’Île-du-Prince-Édouard, 6 phénotypes de virulence parmi les 10 isolats. Comparativement à 2010, il y avait des augmentations quant aux fréquences des isolats virulents au Canada à l’égard des gènes Lr2a, Lr2c, Lr16, Lr26, Lr3ka, Lr11 et Lr30, tandis qu’on a noté une baisse des fréquences des isolats virulents à l’égard des gènes Lr9, Lr24, Lr10 et Lr14a. Lorsqu’un groupe de 161 isolats représentatifs ont été testés sur 5 lignées de plants au stade adulte, aucun isolat n’était virulent à l’égard de Lr22a, la plupart étaient virulents à l’égard de Lr12, Lr13 et Lr37, tandis que seulement 16 étaient virulents à l’égard de Lr35. Lorsque ce même groupe d’isolats a été testé sur 12 lignées additionnelles au stade de semis, aucun isolat n’était virulent à l’égard de Lr19, Lr32, Lr29 et Lr52, tous les isolats étaient virulents à l’égard de Lr15, tandis que leurs réactions variaient à l’égard des gènes Lr2b, Lr3bg, Lr14b, Lr20, Lr23, Lr25 et Lr28. La virulence à l’égard du gène Lr21 a été détectée pour la première fois au Canada chez cinq différents phénotypes de virulence, bien qu’à basse fréquence (3.8%). Ce résultat a des conséquences sur la sélection des lignées de blé au Canada, puisque Lr21 a jusqu’à maintenant été sans faille, et ce, depuis la création du cultivar ‘AC Cora’ en 1994.
Introduction
Wheat leaf rust, caused by Puccinia triticina Eriks. (Anikster et al., Citation1997) (syn. P. recondita Rob. ex Desmaz. f. sp. tritici) is one of the most common and damaging diseases of wheat worldwide (Huerta-Espino et al., Citation2011) and is an annual production concern for wheat growers in Canada. The severity of the disease in Canada changes from year to year but susceptible cultivars are vulnerable to yield losses in most years. Disease severity is driven by a number of factors, including the amount of inoculum coming into Canada from the USA, the ambient temperature, moisture and relative humidity conditions, and the relative level of genetic resistance of the prevalent wheat cultivars, as well as extent of foliar fungicide applications.
Virulence surveys for P. triticina were started in Canada in 1931 and have been conducted annually since that time. This has provided a valuable record of the evolution of virulence and dynamics of the pathogen population (McCallum et al., Citation2016a). The P. triticina population has changed and diversified continuously over this time period (Wang et al., Citation2010), with trends of emerging virulence phenotypes predominating for a number of years, then being replaced by other virulence phenotypes. These changes have important implications for wheat breeding, since new cultivars need to be developed with resistance to this evolving population using combinations of effective resistance genes. The objective of the present research was to survey for virulence phenotypes of P. triticina through sampling of wheat-growing regions conducted across Canada during the growing season of 2011.
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 2011 in various locations throughout Alberta, Manitoba, Saskatchewan, Ontario, Quebec 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 & 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. 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 & 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 & Seto-Goh, Citation2006b). Infection types to all the 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 & 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 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
Virulence on the standard seedling differential lines
In 2011, leaf rust was first observed in southern Manitoba in early June and developed in July and August, reaching 37.5% average flag leaf infection on susceptible wheat lines at the end of the growing season in Manitoba and 6.0% in eastern Saskatchewan (McCallum & Seto-Goh, Citation2012). These were relatively high levels compared with previous years; however, fields in Manitoba were protected from high levels of losses to leaf rust through presence of genetic resistance in the prevalent cultivars, along with foliar fungicide applications. In a broader survey in Saskatchewan, leaf rust was observed in 45% of common wheat fields with an average severity of 5.0% (Fernandez et al., Citation2012). In Ontario, high temperatures and low rainfall during June and July resulted in low incidence of leaf rust, which was only found in 14 of 25 fields surveyed with an average severity of 2.4% (Xue & Chen, Citation2012).
There were 287 single pustule isolates derived from the field samples in 2011, which were analysed for virulence on the 16 standard differentials described above (). These included three from Alberta, 216 from Manitoba and Saskatchewan, 47 from Ontario, 11 from Quebec and 10 from PEI. There were 70 different virulence phenotypes found in Canada during 2011. The most common virulence phenotypes were TDBJ (12.5%), TDBG (10.5%) and MLDS (7.7%). All these races have been fairly common in recent years and were also the most common in 2010 (). Overall, trends are evident over time in the most common virulence phenotypes found in Canada (). This mostly reflects the situation in Manitoba and Saskatchewan, since most samples analysed were collected in these regions. TDBG and TDBJ have been among the most predominant virulence phenotypes in recent years, but although the most frequently found in 2010, MLDS declined in 2011 and other phenotypes such as MFPS and MBTN increased. In 2011, the P. triticina population in Canada was not represented as strongly by a few predominant virulence phenotypes as in previous years, reflected in the relatively higher proportion of ‘other’ virulence phenotypes compared with other years ().
Table 1. Frequency and distribution of virulence phenotypes of Puccinia triticina identified in 2011 by infection types to selected resistance genes.
Table 2. Frequency (%) of predominant P. triticina virulence phenotypes in Canada from 2001 to 2011.
In Alberta, only three isolates were analysed. They were MDPS, MFPS and MNDS (). Both MDPS and MFPS were relatively common in Canada at 5.2% and 6.3% frequency in 2011, respectively, whereas MNDS was only found in Alberta in 2011. These three virulence phenotypes have not been found in recent years from Alberta, but were found previously in Manitoba and Saskatchewan. However, the number of samples from Alberta has been very low.
In Manitoba and Saskatchewan, 33 virulence phenotypes were found from 216 isolates analysed (). The most frequent of these were TDBJ (16.7%), TDBG (13.9%), MLDS and TBBG (both at 9.3%). In 2010, MLDS (29.6%), TDBJ (25.5%) and TDBG (13.8%) were the most commonly found virulence phenotypes from this region. All these phenotypes have been found in this region previously, except for TBBQ, MKDS, TDBS, MFDJ, MJBS, TBBS, TDBQ and TLBG. In the north central region of the USA, bordering Manitoba and Saskatchewan, the predominant virulence phenotypes in 2011 were TBBGJ (14.7%), TDBGG (9.6%) and MLDSD (7.4%) (Kolmer & Hughes, Citation2013), which were similar to the predominant virulence phenotypes in Manitoba and Saskatchewan.
In Ontario, 29 virulence phenotypes were found among 47 isolates tested (). Ontario had nearly as many virulence phenotypes as Manitoba and Saskatchewan (29 vs. 33) even though the number of isolates analysed from Ontario was much lower (47 vs. 216). This reflects the greater degree of diversity in Ontario that has been seen over time. The most frequent phenotypes were MBTN (25.5%), MCTN (8.5%) and then MLDS, TBRK, TBJS, and TCRK (each at 4.3%). Of the virulence phenotypes found in 2011, many were not found in previous years (since 1995) in Ontario, including MCGS, MCRS, MCSS, MFSS, PBDS, PCQQ, TBRF, TBTS, TCGS, TCSS, TDBS, TFRJ, TFRS and TBTK.
From Quebec, 10 virulence phenotypes were identified from among 11 isolates, with MCGS from two isolates and all others represented by one isolate each (). Only TBRK and MCRJ had been found previously in Quebec (since 1995), although there have been less than 10 isolates analysed annually from Quebec over most of these years.
In Prince Edward Island (PEI), there were six virulence phenotypes found among 10 isolates with MCNQ (three isolates), MBPS and MHNQ (two isolates each) being the most common. MCNQ, MHNQ and MCPQ were found in previous surveys of PEI (2006–2010), although the number of isolates analysed were below 20 in each of those years. All of the virulence phenotypes found in PEI in 2011 were unique to this region, except for MBPS. This reflects the unique populations of P. triticina that are found in PEI, and is consistent with findings from previous years.
Virulence on the standard 16 differential lines changed from 2010 (). Figure 1 shows the changes in the frequency of virulent isolates over time for a few of these lines containing genes that are important to Canadian wheat breeding programmes, such as Lr16 and Lr21, or genes for which the frequency of virulent isolates has changed over time, such as Lr2a, Lr9, Lr17 and Lr24. There were slight increases in the frequency of isolates virulent to Lr2a and Lr2c, as T_ _ _ isolates became relatively more frequent than M _ _ _ isolates ( and ), although these are still relatively low frequencies compared with the previous 10 years. There were also increases in the frequency of virulent isolates on Lr16, Lr26, Lr3ka, Lr11 and Lr30, while Lr17 was essentially unchanged. An increase in Lr16 virulence is important to note since it has been near zero in recent years (), and many Canadian cultivars contain Lr16, although the frequency is still low (3.1%). There were decreases in virulence on Lr9, Lr24, Lr10 and Lr14a. The decrease in the frequency of virulence on Lr9 reverses a trend of increasing virulence frequency from 2005 to 2010 ().
Table 3. Frequencies of virulence of Puccinia triticina in Canada in 2011 and 2010 to lines of wheat with single Lr genes for leaf rust resistance.
Fig. 1 Frequency of virulence (%) from 2000–2011a in the Manitoba and Saskatchewan population of P. triticina to near-isogenic lines containing Lr2a, Lr9, Lr16, Lr17, Lr21 or Lr24.
a Data from McCallum & Seto-Goh (2003, 2004, 2005, 2006a, 2006b, 2008, 2009) and McCallum et al. (2010, 2011, 2013, 2016b).
One significant development in 2011 was the detection of virulence for Lr21, for the first time in Canada, at 5.2% of the isolates (). This was found in 14 isolates from Manitoba and one from Saskatchewan. The virulence phenotypes that had some isolates virulent to Lr21 were TDBJ (six isolates out of 36), TDBG (five isolates out of 30), TBBJ (one of three isolates), TDBS (both isolates) and TDBQ. The resistance gene Lr21 has been used extensively in Canada and the USA for leaf rust resistance and is incorporated into many of the current wheat cultivars. Virulence on Lr21 has implications for wheat breeding in Canada, since the gene is now less effective than it was before the evolution of virulence. Virulence to Lr21 was first detected in the USA during 2010 (Kolmer et al., Citation2012) and again in 2011, Lr21 virulence was found in five different virulence phenotypes, mostly in the northern Great Plains of the USA (Kolmer and Hughes, 2013).
Virulence on adult plant differential lines and additional seedling differential lines
When 161 isolates, representing all the virulence phenotypes found from each region, were tested at the adult plant stage on differential lines containing adult plant resistance genes, all isolates were avirulent to Lr22a, but they differed in their reactions to the other differential lines, with high levels of virulence on Lr12, Lr13 and Lr37, and low virulence on Lr35 (), similar to previous years.
Table 4. Infection types of 87 representative isolates from Canada in 2011 tested on near-isogenic lines of ‘Thatcher’ at the seedling (Lr3bg, Lr14b, Lr20, Lr23, Lr25, Lr28) or adult-plant stage (Lr12, Lr13, Lr35 and Lr37).
When this same group of 161 isolates was tested at the seedling stage on 12 additional ‘Thatcher’ differential lines at the seedling stage, all isolates were avirulent to Lr19, Lr32, Lr29 and Lr52 and were virulent to Lr15 while the reaction on Lr2b was similar to Lr2a. All isolates were avirulent to Lr25 except 11–78-1 PBDQ, 11–207-1 PBDG, 11–234-1 PBDG, 11–239-2 PBDG, 11–253-1 PBDG, 11–265-1 PBDG, 11–292-1 PBDQ and 11–317-1 PBDS. Isolates differed in their reactions to Lr3bg, Lr14b, Lr20, Lr23 and Lr28 () although most isolates were virulent on Lr3bg, Lr14b and Lr20. Results for Lr21 confirmed the previous ratings done originally on the smaller sets for all isolates. Only 87 of the 161 isolates tested are shown in as isolates of the same virulence phenotype with the same reaction on the other differentials were represented by a single isolate.
The P. triticina population in Canada originates from inoculum blown northward into Canada from the neighbouring USA. The predominant virulence phenotypes were similar between Manitoba and Saskatchewan and the bordering US north central states, and the virulence phenotypes found in Ontario and Quebec were also similar to phenotypes found in their bordering states of the USA (Kolmer & Hughes, Citation2013). This is consistent with the results found in previous years. There is likely also some selection occurring for virulence in Canada, although the time is relatively short between the arrival of the inoculum from the USA and crop maturity, and therefore the number of asexual generations on which selection can act is limited. Resistance genes Lr1, Lr13, Lr14a, Lr16, Lr21, Lr22a and Lr34 have been used extensively in Canadian wheat cultivars (McCallum et al., Citation2016a). The frequency of virulent isolates is very high for Lr1, Lr13 and Lr14a, while it is low for Lr21 and Lr16, and all isolates tested to date were avirulent to Lr22a and Lr34.
Overall for 2011, the predominant virulence phenotypes found were similar to previous years. The exception was that virulence to Lr21 was found for the first time in Canada and was present in five similar virulence phenotypes. This increase in the frequency of virulence to Lr21 along with the slight increase in virulence to Lr16, are causes of concern for the Canadian wheat crop since both these genes are present in many of the wheat cultivars grown in Canada (McCallum et al., Citation2016a). However, many resistance genes remain fully effective, such as Lr32 and Lr52, and the slow rusting adult plant gene Lr34 continues to provide good levels of resistance, particularly when combined with other resistance genes. The early detection of Lr21 virulence in Canada is a good demonstration of the ability of these broad-scale national surveys to detect new and potentially damaging virulence phenotypes in the pathogen population while they are at a relatively low frequency in the population. This enables the wheat industry to respond in terms of future gene deployment and the creation of more effective combinations of resistance genes in new cultivars.
Acknowledgements
We thank André Comeau, Harpinder Randhawa and Richard Martin who sent samples for analysis, and other cooperators who grew trap rust nurseries.
Related Research Data
References
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