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Article

Macroscopic and Microscopic Phenotyping Using Diverse Yellow Rust Races Increased the Resolution of Seedling and Adult Plant Resistance in Wheat Breeding Lines

by
Kamran Saleem
1,2,
Mogens Støvring Hovmøller
1,
Rodrigo Labouriau
3,
Annemarie Fejer Justesen
1,
Jihad Orabi
4,
Jeppe Reitan Andersen
4 and
Chris Khadgi Sørensen
1,*
1
Department of Agroecology, Aarhus University, 8000 Aarhus, Denmark
2
Plant Protection Division, NIAB, Faisalabad 38000, Pakistan
3
Department of Mathematics, Aarhus University, 8000 Aarhus, Denmark
4
Molecular Breeding Section, Nordic Seed, 8464 Galten, Denmark
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(5), 1062; https://doi.org/10.3390/agronomy12051062
Submission received: 21 March 2022 / Revised: 25 April 2022 / Accepted: 26 April 2022 / Published: 28 April 2022
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Abstract

:
We characterized yellow rust (YR) resistance in sixteen winter wheat breeding lines using three different pathogen races and macroscopic and microscopic phenotyping in lab and greenhouse. Three rust races were used on seedlings and two races on fifth and flag leaf growth stages. The wheat lines were previously characterized to possess none or different quantitative trait loci for YR resistance in field trials. At the seedling stage, twelve lines showed race-specific seedling resistance whereas four lines gave strong seedling resistance to all three races. Seven of eight lines with QTL.1B showed strong seedling resistance against the two races also used at fifth and flag leaves. Microscopic phenotyping of line NOS50906215 (QTL.1B) showed small fungal colonies stopped within 3 dpi associated with extensive hypersensitive response (HR). The lines NOS51014910 and NOS51014911 (QTL.3D alone) showed strong adult plant resistance (APR) from the fifth leaf stage. The lines NOS70140801 and NOS70140808 (QTL.3D + 7B) showed strong APR to one race but partial resistance to the other race at all growth stages. Microscopic phenotyping of line NOS70140801 (QTL.3D + 7B) showed more fungal growth and less HR against the race revealing strong APR compared to the one revealing partial resistance. Line NOS51010312 (QTL.7B alone) showed strong APR response against both races whereas line NOS51010313 (QTL.7B) was susceptible. A partial APR response was observed on line NOS51005019 (no QTLs reported). In conclusion, the approach of combining macroscopic and microscopic phenotyping and diverse pathogen races facilitates the identification of multiple and diverse seedling and adult plant resistance responses to yellow rust in wheat.

1. Introduction

Yellow rust of wheat, caused by the biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), is a serious threat to wheat production worldwide. The global losses caused by the disease is at least 5.5 million tons of wheat annually, equivalent to US$ 1 billion [1]. Widespread yellow rust epidemics occur frequently in major wheat producing countries, causing 5–10% crop losses in many years [2]. Recently, yellow rust of wheat was listed among the top pathogens causing losses higher than 1% globally [3]. Yellow rust was previously prevalent mainly in temperate and maritime wheat growing regions, however, recent devastating epidemics have occurred in warmer areas where the disease was previously infrequent or absent [4]. In recent years, new aggressive races adapted to warmer climates have emerged and spread globally [1,5,6]. Growing resistant wheat varieties may be the most economical and environmental friendly management strategy for yellow rust [7], although fungicide sprays are widely used in intensive production systems in many parts of the world [8]. The European Union (EU) aims to reduce the use of pesticides and especially fungicides were highlighted in the ‘from farm to fork’ strategy of the European Green Deal, which targets a reduction in the use of chemical pesticides by half by 2030 [9]. This can only be achieved by employing crop plants with (durable) disease resistance. Yellow rust resistance can be broadly categorized as seedling resistance and adult plant resistance, the former already detectable at seedling stage but usually expressed during all plant growth stages [10,11]. Seedling resistance is usually effective, but race-specific, so it may be overcome by the evolution of new pathogen races [2,12]. Adult plant resistance (APR) is mainly expressed at adult plant stages and phenotypically, APR can be of major or minor effect relative to disease severity on a susceptible control [13]. APR is generally considered non-race-specific [13,14], however, some APR genes are known to be race-specific, such as Yr11, Yr12, Yr13 and Yr14 [15,16], and more recently, race-specific APR was discovered in French and US wheat varieties [17,18]. If APR is based on multiple genes, it may remain effective for many years even when used over a large area for several years [19,20,21].
To date, 83 yellow rust resistance (Yr) genes have been documented, including 28 APR and 55 seedling resistance genes [22,23,24]. In addition, more than 300 quantitative trait loci (QTLs) associated with yellow rust resistance have been identified in wheat varieties and breeding lines, of which several are associated with resistance at the adult plant stage [25,26,27]. In many wheat varieties, the underlying genetics of QTLs is poorly understood despite correlation with molecular markers and disease phenotype. QTLs displaying larger phenotypic effects relative to the susceptible control have been categorized as “major QTLs”, whereas QTLs associated with minor phenotypic effects were termed “minor QTL” [28]. The combination of several major and minor QTLs, as well as major and minor R-genes, in the same host plant may result in complete resistance [13,29,30]. For instance, the accumulation of four to five minor adult plant resistance genes in wheat lines resulted in a high level of resistance to the yellow rust [29,31].
Tremendous efforts have been made in the identification of loci associated with yellow rust resistance, but the lack of phenotyping platforms discriminating minor individual effects at different growth stages and environments has been a major bottleneck [10,32]. The phenotypic characterization of wheat breeding material is often carried out in field experiments exposed to the prevalent natural pathogen population or inoculation using single races or a mixture of several races [33]. In such cases, the discovery of the phenotypic diversity for resistance in breeding material may be limited [34,35]. The phenotypic effect of seedling resistance acting in a gene-for-gene specific manner may be relatively easily scored and introgressed in wheat breeding germplasm. However, the escalating interest in using adult plant resistance genes represents greater challenges in scoring host responses owing to the need to examine plants at multiple time points during the disease progression as well as the complex influence of environmental factors like temperature and humidity [33,36,37].
Microphenotyping using histological and cytological approaches has the potential to estimate and distinguish the diverse host defense responses from cellular to whole plant level at multiple growth stages [26,38,39,40,41,42]. It has previously been used for the characterization of yellow rust resistance expressed in seedlings and/or at adult plant growth stages, thereby improving the understanding of the underlying resistance mechanisms [38,43,44,45]. Previous studies in microphenotyping of six seedling resistance genes showed distinct spatiotemporal patterns of fungal growth and HR, which clearly reflected their different macroscopic phenotype [42]. Likewise, previous studies on microscopic phenotyping of APR in wheat varieties revealed diverse histological features [26,41,44]. For instance, in wheat cultivar “Kariega” the APR phenotype was strongly associated with cellular lignification and cell death [26], whereas the partial resistance phenotype in the wheat variety “Guardian” was characterized as reduced fungal growth without extensive HR [45]. The histological characterization of APR in the Chinese wheat variety “Xingzi 9104” showed retardation of haustorium and secondary hyphae with extensive HR [43]. The microscopic investigation of yellow rust resistance in the German winter wheat cultivar “Alcedo” showed that combination of two QTLs provide APR phenotype associated with limited cell death however the timing of appearance and the extent of the cell death response varied [39]. Overall, these previous studies highlighted that the APR to yellow rust may involve diverse mechanisms with contrasting histological features.
In the present study, both macroscopic and microscopic phenotyping approaches were applied to explore the diversity of yellow rust resistance in wheat breeding lines originating from a commercial breeding program. These breeding lines were previously included in a genome wide association study to identify QTL for resistance to yellow rust based on field phenotyping. We investigated hypotheses whether additional lab-based macroscopic and microscopic techniques could improve the detection of diverse yellow rust resistance responses in the breeding lines, and whether different categories of macroscopic phenotypes were reflected by distinct histopathological patterns of fungal growth and host responses. Sixteen wheat-breeding lines, proposed to carry up to three QTLs, singly or in combination, were investigated at three plant growth stages (seedling, fifth leaf, and flag leaf). The histopathological characterization was conducted in three of the lines revealing distinct phenotypes depending on growth stage and Pst isolate. The results were discussed with respect to the applicability of improved phenotyping method breeding for yellow rust resistance in wheat.

2. Materials and Methods

2.1. Plant Material, Vernalization, and Growth Conditions

Sixteen winter wheat breeding lines provided by the private breeding company Nordic Seed A/S were used in this study along with susceptible and resistant control wheat varieties (Table 1). The breeding lines were originated from an F6 population and were genotyped with the Illumina 15 K SNPs wheat chip and phenotyped across several years and locations for yield, lodging, and starch content [46] as well as yellow rust disease severity. The lines were selected based on the presence or absence of three putative QTLs conferring resistance to wheat yellow rust. The QTLs had been identified through an association study using SNP molecular markers and field-based disease responses.
The wheat lines were vernalized by placing seeds of each genotype in plastic trays containing wet felt material and blotter paper, after which they were kept in a climate chamber for germination at 17 °C for 5–6 days under photoperiod of 22–27 µmol s−1 m−2 for 12 h. The trays with germinating seedlings were transferred into a climate chamber for 7 weeks of vernalization at 5 °C and a photoperiod of 15–18 µmol s−1 m−2 for 16 h. After vernalization, five seedlings of each line were transferred to 2 L pots containing an organic peat substrate (UNIMULD, Pindstup Mosebrug A/S, Ryomgaard, Denmark). Pots were transferred to spore-proof greenhouse cabins and the temperature was raised gradually to avoid a heat shock effect on plant growth, i.e., 6 °C (night)/10 °C (day) during the first week, 10 °C (night)/14 °C (day) in the second week, followed by 12 °C (night)/18 °C (day) for the remaining experimental period. The light conditions throughout the experiment duration were 16 h light and 8 h darkness, and supplemental light of 200 µmol s−1 m−2 along with 70–80% relative humidity. The three most vigorous plants were kept and trimmed to maintain three main tillers per plant per pot (total 9 tillers/pot). In addition, ten seeds of each line were sown in 7 cm × 7 cm × 7 cm pots in six replications using standard peat-based mix containing slow-release plant nutrients (Pindstrup Mosebrug A/S, Ryomgaard, Denmark) to raise non-vernalized seedlings. These were grown in spore-proof growth cabins, provided with artificial light 50–100 µEm−2 s−1 when daylight <10,000 lux (16 h day/8 h night). The temperature was adjusted to 18 °C day and 12 °C at night and each pot was trimmed after 15 days to maintain eight vigorous seedlings.

2.2. Pathogen Isolates, Multiplication and Growth Conditions

Three Pst isolates representing three named races, Kranich (genetic group PstS8 [47]), Warrior (−) (PstS10) and Boston (PstS0), which were used for the phenotyping of lines at the seedling stage; the former two were further used at 5th leaf and flag leaf growth stages (Table 2). The Pst isolates were retrieved from the stock collection at GRRC (available online: www.wheatrust.org, accessed on 20 March 2022) Denmark, stored in liquid nitrogen (−196 °C) in cryo container, followed by heat shock treatment at 40–42 °C for 2 min. Approximately 5 mg of spores was suspended in 3 mL engineered fluid Novec™ 7100, (3 M, St. Paul, MN, USA) and gently mixed. Standard GRRC differential sets containing 20 wheat differential lines [48] were spray inoculated with the spore suspension using an airbrush spray gun (standard class, RevellGmbH, Bünde, Germany) in a laboratory fume hood to test viability, purity of isolates and race ID. Simultaneously, the seedlings of susceptible wheat cultivars “Anja” and “Morocco” were inoculated for spore multiplication. The seedlings were subsequently sprayed with mist water, and the pots were incubated in a dew chamber at 10–12 °C in darkness for 24 h. After incubation, they were transferred to spore proof greenhouse cabins at 18 °C during day/12 °C during night with a 16 h photoperiod of natural light and supplemental sodium light (100 μmol s−1 m−2) and 8 h dark. Plants were watered automatically with added micronutrients for 10 min every 12 h. The multiplication pots were covered with cellophane bags (Helmut Schmidt Verpackungsfolien GmbH, Königswinter, Germany) prior to sporulation (7 days after inoculation) to prevent cross contamination among isolates. Spores for experimental use were harvested 14–16 days post inoculation (dpi) by shaking the plants inside the cellophane bag, collected in cryo vials, and dried in a desiccator for approximately 3 days before preservation in −80 °C freezer. Differential sets were scored for virulence 17 days after inoculation using a 0–9 scale [48,49] where macroscopic infection types were used to characterize virulence/avirulence based on level of compatibility, i.e., IT 0–6 were considered incompatible (representing avirulence) and IT 7–9 represented a compatible interaction (virulence). The virulence phenotype of the Pst isolates were confirmed and no sign of isolate admixture was observed (Table 2).

2.3. Macroscopic Phenotyping

2.3.1. Seedling Stage

Seedlings of all wheat lines and control varieties were inoculated using a pipette inoculation method [50]. When the second leaf of each seedling was fully developed (15–16 days), it was fixed on acrylic pedestals using surgical tape (Leukofix; BSN Medical GmbH). Eight leaves from each pot were fixed on the acrylic pedestal and three leaves were inoculated with each Pst isolates (Kranich and Warrior (−)). One leaf from each pot was inoculated with the Boston race and one leaf (control) was inoculated with Novec engineered fluid. Five microliter of spore suspension (2 mg/mL of Novec 7100) was applied to the 5 cm central part of each leaf using a pipette. After inoculation, plants were moved to a dew chamber at 10 °C in darkness for 24 h, after which they were transferred to spore proof greenhouse cabins under the same conditions as described earlier. Pots were randomized within six individual cabins wherein each cabin represents one replicate. Disease responses were assessed as infection type (IT) on the second leaf using the 0–9 scale [48,49], after 17 days of inoculation. IT was recorded based on the following phenotypic descriptions: IT 0: no visible disease symptoms, IT 1: minor chlorotic and necrotic flecks, IT 2: chlorotic and necrotic flecks without sporulation, IT 3: chlorotic and necrotic areas with few minuscule pustules, IT 4: chlorotic and necrotic areas with limited sporulation, IT 5–6: chlorotic and necrotic areas with intermediate or moderate sporulation, IT 7: abundant sporulation with moderate chlorosis, IT 8–9: abundant and dense sporulation with insignificant or no chlorosis and necrosis.

2.3.2. Adult Plants

Six pots of all wheat genotypes were inoculated at stem elongation stage (GS 32–34) when the fifth leaf was fully developed while another six pots were inoculated at flag leaf stage (GS 47–49) [51]. For inoculation, a fresh urediniospores suspension (4 mg/mL of Novec 7100) was prepared for each Pst isolate. The spore suspension was applied to a 5 cm section of the adaxial surface of a leaf, marked by a permanent marker, using a fine camelhair paintbrush (size 1). The entire marked leaf area was uniformly brushed until the inoculated leaf portion became wet without run-off. The leaves of six tillers (3 tillers/Plant) in each pot was inoculated with two Pst isolates (three tillers/Pst isolate) while three tillers as control with Novec 7100. In total, 18 leaves of each genotype were inoculated with an individual Pst isolate and assessed for macroscopic and microscopic phenotypes. After inoculation, plants were mist sprayed with distilled water and incubated in a dark dew chamber at 10–12 °C and 100% relative humidity for 24 h to ensure infection. After incubation, inoculated plants were transferred to spore-proof greenhouse cabins under the conditions described above. Plants were randomized between six cabins in a way that one pot of each wheat genotype was represented in each cabin (block). Disease assessment was carried out by recording infection type (IT) on the 0–9 scale 19 days after inoculation.

2.4. Microscopic Phenotyping

2.4.1. Sample Collection

Four leaves (one leaf per pot) were collected at 3, 6, and 9 days post inoculation from each wheat line at seedling, fifth leaf, and flag leaf growth stages. The inoculated leaf sections, including an additional 1 cm above and below the marked area, were detached. The remaining six inoculated leaves were used for assessment of IT on the 0–9 scale and macroscopic imaging using a Canon EOS 7D digital SLR camera equipped with a Canon macro EF 100 mm f/2.8 L IS USM lens (Canon, Tokyo, Japan).

2.4.2. Fixation, Staining, and Microscopy

Leaf segments for microscopy were collected in 15 mL falcon tubes (Corning Science, Mexico) and immediately covered with fixation solution. Leaf samples were fixed and stained according to Moldenhauer et al. 2006 [38] with minor modification. In brief, leaf samples were fixed in ethanol: chloroform (3:1, v/v) + 0.15% (v/w) trichloroacetic acid solution for at least 24 h. For the complete removal of chlorophyll, the solution in each tube was changed and fresh solution was added after 24 h. To remove chloroform, leaf segments were washed twice in 50% ethanol for 15 min and then 0.05 M NaOH was added. For seedlings, samples were kept in this solution for 30 min while for adult plant samples, leaves were incubated at 95 °C in 0.05 M NaOH for 50 min. Leaf samples were then washed three times with deionized water (DI). Afterwards, 0.1 M Tris–HCl buffer (pH 8.5) was added in Falcon tubes which were incubated in a fume hood for 30 min. The leaf samples were stained in 0.1% (w/v) Calcoflour White M2R (dissolved in 0.1 M Tris-HCl Buffer) for 5 min (Rohringer, 1977, Kilb, Austria). This was followed by washing four times with deionized water (10 min each) and once with 25% glycerol for 30 min. Finally, samples were incubated overnight in DI. Leaf samples were stored in 50% glycerol until use for microscopy.
For microscopy, leaf segments were mounted on a glass slide containing 75% glycerol. Colony size (CS) and the extent of hypersensitive response (HR) was measured with a Leica Laborlux S Microscope (Leitz Wetzlar, Germany) equipped with optics for epifluorescence. Fungal colonies and host autofluorescence were visualized using a UV-D filter with excitation filter setting 355–425 nm and barrier filter LP 470 nm. Thirty colonies were observed at each time point across all leaf segments and treatments. Colony dimensions were measured with a calibrated eyepiece micrometer and the size was calculated as largest length × largest width × π/4 [52]. The extent of HR was measured as a percentage of the area showing autofluorescence around the fungal colony and description of quantitative measurements of HR is given in Supplementary Figure S1. Images of representative colonies were recorded using OLYMPUS FV1200 confocal laser scanning microscope. Leaf tissues and fungal structures were excited with laser beams of 405 nm and 515 nm lasers and two channels with filter setting 435–515 nm and 530–625 nm detected emitted light signals respectively. Layers of confocal planes with 0.75 μm separation were collected for Z-stacks. IMARIS® bitplane 6.2 software (Oxford, UK) was used to merge the images of two channels and execute 3D-projections. All resulting 2D images were adjusted for brightness and contrast to obtain an equal representation of the background signal from the healthy plant tissue using the free software paint.net (available online: https://www.getpaint.net/, accessed on 20 March 2022).

2.5. Statistical Analysis

The statistical analyses were performed using the R-software (R Core Team, 2018). The comparisons between the mean colony sizes and the HR among the three wheat lines for each observation time points (dpi) were made by permutation tests, and the reported confidence intervals were constructed using non-parametric bootstrap (with 10,000 bootstrap samples) implemented in the R-package “postHoc” [53]. The p-values were adjusted for multiple comparisons by the False Discovery Rate (fdr) method [54].

3. Results

3.1. Macroscopic Phenotyping

The experimental setup provided consistent and high-resolution phenotyping results, which enabled us to detect distinct categories of disease resistance responses in diverse winter wheat breeding lines. The macroscopic infection types (ITs) were consistent across replications for all combinations of host lines and growth stages and individual Pst races (Figure 1). Images of representative macroscopic phenotypic responses for all host-pathogen combinations are shown in Figure 2.
Overall, Pst resistances with major effects as well as minor effects were observed within the investigated breeding lines, and race-specificity was detected at both seedling stage and adult plant stages. Seven of eight wheat lines proposed to carry QTL-1B (lines 1–8, Figure 2), either singly or in combination, showed seedling resistance which was effective at all plant growth stages against the isolates of two genetically different races, Kranich and Warrior (−). In contrast, the two races resulted in distinctly different phenotypes on wheat line 9 and 10 carrying QTL combination 3D + 7B. Both lines displayed APR with major effect to the Kranich race, but resistance with minor effect to Warrior (−) at all three growth stages. Wheat line 11 and 12 with QTL-3D alone showed APR to both races, which was expressed already at the fifth leaf stage and fully developed at the flag leaf stage. A large phenotypic difference was observed between two sister lines (line 13, 14) supposed to carry QTL-7B alone, suggesting recombination between genetic markers and the resistance locus. The two Pst races displayed contrasting phenotypes for line 13 (QTL-7B), showing APR to the Kranich race and seedling resistance to Warrior (−) race. An unexpectedly high (compatible) IT was observed in line 2, which was supposed to carry QTLs 1B + 3D + 7B and in line 14, supposed to carry QTL-7B; this was the case for both Pst races. Line 15 and 16 with no previously identified QTLs showed a similar response to both races. Intermediate level of APR was observed in line 16 whereas line 15 was susceptible.
An additional Yr15-virulent race (termed “Boston”) was used at the seedling stage (Supplementary Figure S2). Inoculation with the Boston race revealed additional seedling resistance in thirteen of the wheat lines. These included the lines 9–14 supposed to carry QTL-3D or QTL-7B as well as line 15 and 16 without previously identified QTLs. Line 1, 3, and 6 with QTL-1B had an intermediate IT similar to Avocet/Yr15 and variety “Mariboss”, providing support for the presence of Yr15 in these lines. The other five lines supposed to carry QTL-1B, had additional seedling resistance against the Boston race, revealed by a low IT. The control varieties “Cartago” and “Anja” showed the expected high infection types. Overall, the phenotyping approaches under controlled experimental conditions at multiple growth stages and against diverse Pst races resolved high level of diversity of Pst resistance in 16 breeding lines.

3.2. Microscopic Phenotyping

Three of the sixteen lines showing contrasting macroscopic phenotypes for combinations of growth stage and race were characterized histologically. Line 7 (QTL-1B) represented a resistance with major effect at both seedling and adult plant growth stages, line 9 (QTL-3D + 7B) showed resistance with minor effect at all growth stages to the Warrior (−) race and APR to the Kranich race, and line 15 (no identified QTLs) represented a susceptible phenotype (Figure 2).
The histological results for these three wheat lines showed a distinct growth pattern of fungal colony size and associated HR. The mean colony size and mean HR showed significant differences with respect to time point after inoculation, growth stage and Pst race (Figure 3, Supplementary Table S1). Macroscopically, wheat line 7 (QTL-1B) reflected resistance with major effect against both Pst races at seedling, fifth leaf and flag leaf stages. Microscopically, wheat line 7 (QTL-1B) showed mostly small colonies with high levels of HR coverage at all three growth stages when infected with the Kranich race, although few small colonies with low HR encasement were also observed in seedlings at 3 dpi. The patterns of colony sizes and HR in seedlings were different for the Warrior (−) race, where relatively larger colonies with high HR coverage were observed at all time points, but in particular at 9 dpi. At fifth leaf and flag leaf stages, fungal growth and HR were mostly similar for both Pst races.
The differences in colony morphology and associated HR in line 7 were further investigated by confocal laser scanning microscopy. The histological investigation revealed that fungal colony morphology and extent of HR were different at the seedling stage for the two Pst races (Figure 4). All colonies in incompatible interactions involving the Kranich race were small, developing sub-stomatal vesicles (SSV), primary hyphae (PH), and haustorial mother cells (HMC) encased by autofluorescent host cells without any sign of haustorial bodies. The host autofluorescence appeared at an early stage of the infection and covered most of the fungal structures. For many colonies, autofluorescence was not only limited to cells with direct contact with fungal structures, adjacent host cells were often also affected. Many incompatible Warrior (−) colonies developed secondary hyphae at 3 dpi with large portions covered with autofluorescent host cells. The mean colony size increased many folds by developing additional runner hyphae, but at 9 dpi these were completely encased with extensive HR. At fifth and flag leaf stages, colony morphology and level of HR were similar for both Pst races. The macroscopic and microscopic characteristics of wheat line 7 are consistent with a typical qualitative seedling resistance response.
The wheat line 9 (QTLs 3D + 7B), which at the macroscopic level showed strong APR to the Kranich race and partial resistance to Warrior (−) race at all growth stages, exhibited significant difference in mean colony size and HR with respect to time point and growth stages at the microscopic level (Figure 3). At the seedling stage, the Kranich race resulted in increasing colony sizes with time, associated with low HR coverage. The colonies were large and intermingled at 9 dpi, and it was not possible to distinguish and measure the size of individual colonies. At the fifth leaf stage, most colonies were small and associated with 60–75% mean HR coverage at 3 and 6 dpi. At 9 dpi, most of the colonies had circumvented the HR and colony sizes had significantly increased despite the presence of high levels of HR. The mean colony size of the Kranich race was smaller in the flag leaf compared to seedling and fifth leaf. In the flag leaf small colonies with low HR coverage were seen at 3 dpi but afterwards at 6 and 9 dpi most colonies had about a 50% HR coverage. At 9 dpi, most of the colonies were bigger than at 6 dpi but without any significant change in the level of mean HR coverage. Overall, the mean colony sizes were smaller in flag leaves compared to earlier growth stages while HR coverage increased from seedling to fifth leaf stage and at 6 and 9 dpi in the flag leaves. This pattern suggested that, in case of the APR phenotype, colonies often circumvented HR to continue growth.
The partial seedling resistance response of wheat line 9 against Warrior (−) revealed large colonies with extensive HR encasement (Figure 3). At 3 dpi, colonies were small with low HR coverage (25% mean HR), but afterwards colony size and HR increased significantly with time and relatively large colonies with 75% HR coverage were observed at 9 dpi. The most significant shift in mean colony size was observed from 6 to 9 dpi without significant change in the level of HR coverage at seedling stage. The partial resistance at fifth leaf stage against Warrior (−) was characterized as relatively larger colonies than in seedling at 3 and 6 dpi, however at 9 dpi mean colony size was comparatively smaller. Both mean colony size and HR significantly increased from 3 to 9 dpi. In flag leaves, temporal increase in colony size and HR was observed but the mean colony size and HR were smaller at flag leaf stage than in seedling and fifth leaves. In comparison to Kranich, the mean colony size in flag leaves at 9 dpi was smaller whereas mean HR coverage was significantly higher in case of Warrior (−) (Supplementary Table S1). Overall, the level of HR was highly dependent upon the growth stage and time point after inoculation.
Colony morphology and associated host HR in line 9 were characterized with confocal microscopy, demonstrating that the two races resulted in distinctly different histological features (Figure 5), which generally confirmed the differences described above. At the seedling stage, the Kranich race resulted in colonies with secondary and runner hyphae at 6 dpi, increasing many folds in size at 9 dpi with weak signs of HR. At flag leaf stage, most of the colonies develop secondary hyphae at 6 dpi largely surrounded by autofluorescent host cells whereas many colonies developed runner hyphae at 9 dpi along with patches of autofluorescent host cells. The colony growth at seedling stage was comparatively less at both 6 and 9 dpi against Warrior (−). Most of the colonies develop secondary hyphae completely encased with extensive HR at 6 dpi. Despite the extensive HR, colonies of Warrior (−) developed many runner hyphae at 9 dpi at seedling stage. At the flag leaf stage, colony morphology and host autofluorescence were mostly similar for both Pst races. The colony growth was longitudinal between the veins and host autofluorescence appeared in patches along the secondary and runner hyphae. The HR level at 9 dpi was slightly higher against Warrior (−) than Kranich. Overall, the microscopic phenotype and histological features of line 9 against the Kranich race showed a reduced colony size in flag leaves compared to seedling and fifth leaves, and a time point dependent HR expression in the APR response. The all-stage partial resistance of line 9 against Warrior (−) was manifested as a reduction in colony size with growth stage but similar HR expression relative to colony size at all growth stage.
Wheat line 15 (without identified QTLs) represented a compatible interaction with both races while at microscopic scale, it showed that most of the colonies continued their growth without HR during all growth stages against both Pst races although cell death was occasionally observed (Figure 3). The histological observation of representative colonies by confocal microscopy revealed many secondary and runner hyphae at 6 dpi (both races), and at 9 dpi all colonies were large, intermingled, and appeared as a mycelial network during all three growth stages (Supplementary Figure S3).

4. Discussion

In a greenhouse study we investigated resistance to yellow rust in sixteen wheat lines from a breeding program at the private Danish breeding company Nordic Seed A/S using both macroscopic and microscopic phenotyping approaches. The wheat lines had previously been characterized to carry different QTLs for yellow rust resistance through association mapping and disease assessment in field trials. We inoculated with three genetically diverse yellow rust races at the seedling stage and two of these races were also used at fifth and flag leaf stage. Overall, we were able to enhance the resolution of yellow rust resistance responses in the material, which revealed distinct categories of contrasting resistance phenotypes. Macroscopically, resistance with major effects at both seedling and adult plant stages, as well as resistance with minor effects, were observed among the lines. Microscopically, we observed race-specific seedling resistance characterized by small fungal colonies strongly encased by hypersensitive response (HR). Wheat line 9, which had strong APR to the Kranich race but partial effect against the Warrior (−) race at all growth stages, showed common histological features to both races at the flag leaf stage. The histological observations in line 9 suggest that APR may involve additional mechanisms than typical HR response.
Among the sixteen wheat breeding lines, seven lines with QTL-1B, either single or in combination with other QTLs, showed major resistance effects that was expressed at all growth stages against the Kranich and Warrior (−) races, which suggests that QTL 1B may contain one or more major seedling resistance gene(s). It has been previously documented that chromosome 1B contains numerous yellow rust resistance QTLs or genes with major effects, including Yr9, Yr10, Yr15, Yr24, Yr26, Yr64, and Yr65 [55]. The intermediate seedling infection type revealed by the Yr15 virulent Boston race in three lines (1, 3, and 6) with QTL-1B suggests that QTL-1B may contain Yr15. This was further supported by microscopic observations in line 7 (QTL-1B) inoculated with the Warrior (−) and Kranich races, revealing only small fungal colonies encased with extensive HR triggered at early stages of infection without haustorial bodies. These histological features were consistent with our previous histological characterization of AvocetYr15 in which small fungal colonies with extensive HR was observed against an avirulent Pst isolate [42]. We also observed that the colony size and level of HR varied significantly between the Kranich and Warrior (−) at the seedling stage despite a clearly incompatible infection type. There is a possibility that Warrior (−) partly suppress the effect of Yr15 or is recognized during a later stage of infection. Another explanation could be heterozygosity for Yr15-virulence in the Warrior (−) race. Wheat line 2 assigned QTL 1B showed a compatible phenotype against Kranich and Warrior (−), suggesting that it does not contain Yr15. Inoculation of line 2 with the Yr15 virulent Boston race revealed an incompatible phenotype which postulates additional seedling resistance not detected by Kranich and Warrior (−). The Boston race further revealed additional seedling resistance in the lines 4, 5, 7, and 8 with QTL 1B, as well as in all lines showing seedling susceptibility to Kranich and Warrior (−).
Phenotyping of the breeding lines carrying either QTL-3D, QTL-7B or the combination of both QTL, revealed APR with major and minor effect at fifth and flag leaves, but depending on the Pst race. The lines with QTL-3D showed a seedling susceptibility and gave a complete resistance to both races in the flag leaf suggesting that QTL-3D loci may contain one or more APR gene(s). Five QTLs are known on wheat chromosome 3D. These include QYr.tam-3D, Quaiu, QYr-3DS, QYR4, QYr.cim-3D and QYr.inra-3DS characterized in wheat varieties “Quaiu3”, “Opata85”, “Chapio”, and “Recital”, respectively [14,56,57,58]. In addition, two APR genes, Yr49 and Yr71, were also reported on chromosome 3D [33]. In most of these studies, the QTLs were identified based on association between mapped DNA makers and resistance phenotypes under field conditions. Microscopic phenotyping of two wheat lines with QTL-3D was executed twice in our study, but due to the failure in recording colony size and HR, further studies are needed to understand the exact features of this particular APR phenotype. For the future study, it could be informative to investigate the development in fungal growth from seedling to fifth and flag leaf stages and association of this APR phenotype with host HR. Contrasting phenotypic responses were observed with respect to line ID and Pst race for the two wheat sister lines proposed to carry only QTL-7B. One sister line with QTL-7B alone showed APR to the Kranich race, but seedling resistance to Warrior (−). The results suggest the presence of at least two different resistance genes in this wheat line, identified by Kranich and Warrior (−). Three APR genes; Yr39, Yr52 and Yr59 and several QTLs were previously identified on chromosome 7B [59,60,61,62]. The other sister line proposed to carry QTL-7B was susceptible to both races, suggesting recombination between marker(s) and the resistance locus. Recombination might also explain the susceptible phenotype of one of the wheat lines expected to possess the three QTLs 1B, 3D, and 7B.
Several previous studies have shown that the combination of several APR genes (major/minor) and/or QTLs may be the most appropriate approach to attain high level and increased durability of yellow rust resistance compared to major monogenic resistance [29,31,63]. The genetic interactions among components of APR resistance could be postulated as additive or epistatic by visualizing their phenotype. In the current study, the resistance showed by the wheat lines having the two QTLs (3D + 7B) were less effective than the resistance of line carrying only QTL-3D. One explanation could be that the QTL-3D may carry an APR gene, which is less effective in the presence of QTL-7B. The different responses of these two lines to the Warrior (−) race are difficult to interpret due to the identification of additional resistance from the seedling stage and up to the flag leaf stage.
The histological investigations of line 9 carrying two QTLs (3D + 7B) were used to explore to which extent the micro-phenotypes reflected their contrasting macroscopic infection types with respect to Pst races i.e., strong APR against the Kranich and all-stage partial resistance against the Warrior (−) race. Despite a stronger macroscopic phenotype in the flag leaf, colonies of Kranich were generally larger and with less HR than for the Warrior (−) race. The pattern might suggest that the APR against Kranich dependents on other resistance mechanisms in addition to HR. Previous histological characterizations of the APR genes Yr18 and Yr29 revealed that these were not significantly associated with HR in the host [64,65]. Other previous studies reporting diverse micro-phenotypes of APR in wheat varieties, support our findings. Microscopic phenotyping of APR in South African bread wheat cultivar “Kariega” governed by two major and two minor QTLs [66] revealed that one major QTL was associated with lignification without restriction of fungal growth while a second major QTL did not show lignified host tissue, but fungal growth was restricted [44]. The partial resistance in the UK wheat cultivar “Guardian” was governed by the combination of three QTLs [67] and none of these were associated with extensive host cell death [45]. These results revealed that in addition to lignification and HR, the simultaneous and possibly synergistic induction of other resistance reactions may be involved in APR. For a better understanding of the APR mechanisms, one may consider other parameters for microscopic phenotyping, including the number of colonies per unit area, haustorial development, and cellular lignification. Further studies are needed to elaborate if the APR induced by the Kranich race is also involved in the response against the Warrior (−) race, or if the effect against the Warrior (−) race is solely due to the identified seedling resistance.
The resistance phenotypes of APR gene like Yr18 and Yr29 are sensitive to environmental factors, in particular the temperature [68]. For instance, the APR genes Yr18 and Yr36 provide partial resistance, but their responses were temperature sensitive [69,70,71]. In the current study, many lines including those with QTL-7B, QTL-1B + 3D + 7B, and susceptible varieties showed necrosis at fifth leaf stage, most pronounced for the Warrior (−) race which could reflect sensitivity to the environmental conditions at the fifth leaf stage. A standard temperature and relative humidity were maintained throughout the experiment but wheat lines with extensive necrosis might be sensitive to shifts in day and night temperature, and in light under greenhouse conditions. Precise characterization of necrotic phenotypes of such wheat lines is challenging as specific low and high temperature screens should be completed. Another explanation could be that the fifth leaves were more sensitive to the inoculation method. The role of leaf age, leaf position, and spore concentration on phenotyping for yellow rust resistance cannot be excluded.

5. Conclusions

In many breeding programs, the breeding material is tested under field condition at adult plant stages without a seedling test. Under such conditions, it is hard to infer whether the resistance is conferred by seedling resistance genes or APR genes. In our study, the distinct categories of seedling resistance in the same breeding population enforced that phenotyping of wheat breeding lines against multiple Pst races greatly improve the resolution of potential host resistances involved. The seedling phenotypes of breeding lines also illustrate that seedling virulent Pst races is a prerequisite for the identification of APR in breeding material. The macroscopic and microscopic phenotyping approaches identified different categories of resistance responses and variability, which to a large extent depended on Pst race and plant growth stage. Macroscopic phenotyping differentiated wheat lines showing seedling resistance from those showing additional adult plant resistance both with major and minor effects. We observed some cases with similarity between microscopic phenotypes on seedlings and adult plants, and other cases where the extent and efficacy of resistance responses were different. The study suggests that a high level of resistance against yellow rust may be achieved by using wheat lines with a combination of QTL-1B and QTL-3D. It provides evidence of the presence of seedling resistance or APR in associated genetic loci, which can be further narrowed down to the candidate genes. Increased durability may be achieved if diverse and multiple sources of resistance operational at different stages of infections are used.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/agronomy12051062/s1, Figure S1: Scale for HR observation as an area of host tissue in percentage around the fungal structures showing autofluorescence. Infected leaf samples were stained with Uvitex 2B and visualized under epifluorescence microscope for recording HR around fungal colonies. (A) HR is categorized as 0 when fungal colonies grow without any sign of host autofluorescence. (B) HR is recorded up to 25% if HR is apparent around one hyphae of fungal colony. (C) Up to 50% HR is recorded when autofluorescence is expressed along one side of colony. (D) HR is 51–75% when most of the fungal colony is covered with host autofluorescence. (E) HR is recorded as 76–100% if fungal colony is growing bigger while HR also increases and covered most of the colony. (F) Fungal colony is completely surrounded by HR and assigned as 100%. Figure S2: The phenotypic reaction of 20 wheat lines at seedling stage against Kranich, Warrior (−), Kalmar, and Boston (considered Yr15 virulent) races of Pst recorded at 17 days post inoculation. Figure S3: The microscopic phenotype of wheat line 15 (control) having no identified QTL at seedling, fifth leaf and flag leaf stages recorded at 9 dpi against Kranich and Warrior (−) Pst races. All colonies were large, intermingled and with highly branched mycelium net. Scale bar = 100 µm. Table S1: Variation in colony sizes and HR in three QTLs lines against two Pst races at three growth stages.

Author Contributions

Conceptualization, C.K.S., K.S., M.S.H. and A.F.J.; methodology, C.K.S., K.S. and M.S.H.; writing—original draft preparation, K.S. and R.L.; writing—review and editing, K.S., C.K.S., M.S.H., A.F.J., J.O. and J.R.A.; visualization, K.S.; formal analysis, R.L., K.S. and C.K.S.; investigation, K.S.; resources, J.O., J.R.A. and M.S.H.; supervision, M.S.H. and C.K.S.; project administration, M.S.H.; funding acquisition, M.S.H., A.F.J., J.O. and J.R.A. All authors have read and agreed to the published version of the manuscript.

Funding

This work is part of a PhD project funded by innovation fund Denmark, Ministry of Higher education and science (Grant No. 19052, MULTIRES) and Aarhus University.

Institutional Review Board Statement

Not applicable for studies not involving human or animals.

Informed Consent Statement

Not applicable for studies not involving human.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are highly grateful to Ellen Jørgensen, Janne Holm Hansen, and Steen Meier, Aarhus University, for technical assistance during experiments. Brent McCallum, Agriculture and Agri-Food Canada is highly acknowledged for his critical review of this manuscript.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. The infection type of 16 wheat breeding lines along with three control wheat varieties at seedling, fifth leaf and flag leaf stages against Kranich and Warrior (−) races. Infection type was recorded using 0–9 scale [49] (McNeal et al., 1971). The box represents the variation in infection type across six replicates. The dots represent the observation, which is outside the interquartile range of the boxplot.
Figure 1. The infection type of 16 wheat breeding lines along with three control wheat varieties at seedling, fifth leaf and flag leaf stages against Kranich and Warrior (−) races. Infection type was recorded using 0–9 scale [49] (McNeal et al., 1971). The box represents the variation in infection type across six replicates. The dots represent the observation, which is outside the interquartile range of the boxplot.
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Figure 2. The macroscopic phenotype of 16 wheat breeding lines and control wheat varieties at seedling, fifth leaf, and flag leaf stage against Kranich (A) and Warrior (−) (B) races of Pst. seeding, and adult plant resistances phenotypes were identified in breeding lines. The control wheat varieties showed expected phenotype. QTL position and number of QTL were given within parenthesis.
Figure 2. The macroscopic phenotype of 16 wheat breeding lines and control wheat varieties at seedling, fifth leaf, and flag leaf stage against Kranich (A) and Warrior (−) (B) races of Pst. seeding, and adult plant resistances phenotypes were identified in breeding lines. The control wheat varieties showed expected phenotype. QTL position and number of QTL were given within parenthesis.
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Figure 3. Mean colony sizes, HR coverage, and their variation with respect to growth stage in three wheat lines against Kranich and Warrior (−) races at 3, 6, and 9 days post inoculation. The mean colony size and HR at individual time point followed by same letter are not significantly different (p < 0.05).
Figure 3. Mean colony sizes, HR coverage, and their variation with respect to growth stage in three wheat lines against Kranich and Warrior (−) races at 3, 6, and 9 days post inoculation. The mean colony size and HR at individual time point followed by same letter are not significantly different (p < 0.05).
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Figure 4. The fungal structures and host autofluorescence in wheat line 7 (1B) recorded at 3 and 9 dpi against Kranich (A,B) and Warrior (−) race (C,D) at seedling stage. Basal fungal colony structures include germ tube (GT) which penetrate through stomata (S) and develop into sub stomatal vesicle (SSV). SSV develop to two primary infection hyphae (PH) and haustorial mother cells (HMC). HMC develop into haustoria (H), secondary hyphae (SH), and runner hyphae (RH). Extensive autofluorescence (AF), as an indicative of hypersensitive response (HR) is seen in close contact with fungal structures Scale bar = 30 µm.
Figure 4. The fungal structures and host autofluorescence in wheat line 7 (1B) recorded at 3 and 9 dpi against Kranich (A,B) and Warrior (−) race (C,D) at seedling stage. Basal fungal colony structures include germ tube (GT) which penetrate through stomata (S) and develop into sub stomatal vesicle (SSV). SSV develop to two primary infection hyphae (PH) and haustorial mother cells (HMC). HMC develop into haustoria (H), secondary hyphae (SH), and runner hyphae (RH). Extensive autofluorescence (AF), as an indicative of hypersensitive response (HR) is seen in close contact with fungal structures Scale bar = 30 µm.
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Figure 5. Fungal colony morphology and host autofluorescence of wheat line 9 (3D + 7B). Fungal colonies and host autofluorescence at seedling stage and flag leaf stage recorded at 6 and 9 dpi. The Kranich race (A) is revealing APR, and the Warrior (−) race (B) is revealing partial resistance at both growth stages. GT: germ tube (GT), S: stomata, SSV: Sub stomatal vesicle (SSV). PH: primary infection hyphae, HMC: Haustorial mother cells, H: Haustoria, SH: Secondary hyphae and RH: Runner hyphae. AF: Autofluorescence (AF), as an indicative of hypersensitive response (HR). Scale bar = 30 µm.
Figure 5. Fungal colony morphology and host autofluorescence of wheat line 9 (3D + 7B). Fungal colonies and host autofluorescence at seedling stage and flag leaf stage recorded at 6 and 9 dpi. The Kranich race (A) is revealing APR, and the Warrior (−) race (B) is revealing partial resistance at both growth stages. GT: germ tube (GT), S: stomata, SSV: Sub stomatal vesicle (SSV). PH: primary infection hyphae, HMC: Haustorial mother cells, H: Haustoria, SH: Secondary hyphae and RH: Runner hyphae. AF: Autofluorescence (AF), as an indicative of hypersensitive response (HR). Scale bar = 30 µm.
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Table
Table 1. Wheat breeding and control lines used for phenotypic characterization of Pst resistance.
Table 1. Wheat breeding and control lines used for phenotypic characterization of Pst resistance.
Wheat LineLine IDNo. QTLQTL Chromosome Location aField Responses b
Year-IYear-II
1NOS-5090481431B7B3D91
2NOS-5090482331B7B3D95
3NOS-180090121B-3D12
4NOS-180721821B-3D22
5NOS-170580421B7B-11
6NOS-170580821B7B-11
7NOS-5090621511B--22
8NOS-5090621611B--11
9NOS-701408012-7B3D54
10NOS-701408082-7B3D65
11NOS-510149101--3D21
12NOS-510149111--3D21
13NOS-510103121-7B-53
14NOS-510103131-7B-43
15NOS-51005012None---45
16NOS-51005019None---44
17CartagoSusceptible Control
18AnjaSusceptible Control
19MaribossResistant Control
20AvocetYr15 (seedling test)Resistant Control
a “-” indicates that the QTL on 1B, 7B and/or 3D were not previously identified in the wheat line based on SNP marker analysis. b Field responses on a 1–9 disease severity scale. 1: 0 percent disease; 2: 0.1 percent; 3: 0.5 percent; 4: 1 percent; 5: 5 percent; 6: 10 percent; 7: 25 percent; 8: 50 percent and 9: >75 percent.
Table
Table 2. Puccinia striiformis isolates used for the phenotyping of wheat breeding lines. Pst isolates with their ID, common name, origin and their phenotypic response on standard differential sets of 20 wheat lines, representing 18 different yellow rust resistance genes (Yr).
Table 2. Puccinia striiformis isolates used for the phenotyping of wheat breeding lines. Pst isolates with their ID, common name, origin and their phenotypic response on standard differential sets of 20 wheat lines, representing 18 different yellow rust resistance genes (Yr).
Isolates IDCommon Race NameOriginVirulence for Yr Genes a
123456789101517252732AvSAmbSp
DK02d/12KranichDenmark123--6789--1725-32AvSAmb-
DK110/15Warrior (−)Denmark1234-67-9--1725-32AvS-Sp
DK92/02BostonDenmark123-----9-15(17)25--AvS--
a Avirulence is indicated by “-” while virulence was indicated by the corresponding Yr-gene or cultivar (Ambition (Amb)).
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Saleem, K.; Hovmøller, M.S.; Labouriau, R.; Justesen, A.F.; Orabi, J.; Andersen, J.R.; Sørensen, C.K. Macroscopic and Microscopic Phenotyping Using Diverse Yellow Rust Races Increased the Resolution of Seedling and Adult Plant Resistance in Wheat Breeding Lines. Agronomy 2022, 12, 1062. https://doi.org/10.3390/agronomy12051062

AMA Style

Saleem K, Hovmøller MS, Labouriau R, Justesen AF, Orabi J, Andersen JR, Sørensen CK. Macroscopic and Microscopic Phenotyping Using Diverse Yellow Rust Races Increased the Resolution of Seedling and Adult Plant Resistance in Wheat Breeding Lines. Agronomy. 2022; 12(5):1062. https://doi.org/10.3390/agronomy12051062

Chicago/Turabian Style

Saleem, Kamran, Mogens Støvring Hovmøller, Rodrigo Labouriau, Annemarie Fejer Justesen, Jihad Orabi, Jeppe Reitan Andersen, and Chris Khadgi Sørensen. 2022. "Macroscopic and Microscopic Phenotyping Using Diverse Yellow Rust Races Increased the Resolution of Seedling and Adult Plant Resistance in Wheat Breeding Lines" Agronomy 12, no. 5: 1062. https://doi.org/10.3390/agronomy12051062

APA Style

Saleem, K., Hovmøller, M. S., Labouriau, R., Justesen, A. F., Orabi, J., Andersen, J. R., & Sørensen, C. K. (2022). Macroscopic and Microscopic Phenotyping Using Diverse Yellow Rust Races Increased the Resolution of Seedling and Adult Plant Resistance in Wheat Breeding Lines. Agronomy, 12(5), 1062. https://doi.org/10.3390/agronomy12051062

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