Evaluation of Pea Accessions Differing in Flower and Seed Coat Pigmentation for Resistance to Fusarium avenaceum Root Rot

Abstract

Pea production across the world is significantly limited by root rot disease, which is caused by many fungal and oomycetes pathogens. In Canada, Fusarium avenaceum is the most devastating pathogen of the Fusarium root rot complex of pea. Host genetic resistance is the most effective control method for this disease. Evaluation of global pea accessions and Canadian varieties for F. avenaceum root rot resistance has not been reported to date. This study evaluated 20 pea accessions of different market classes with pigmented or nonpigmented seed coats and flowers for F. avenaceum resistance under controlled conditions. The pea accessions CDC Acer, CDC Vienna, PBA OURA, Morgan, CDC Blazer, CDC Dakota, and PI 280609, which have pigmented flowers and seed coats, were identified as resistant or partially resistant to F. avenaceum. This was based on their root rot severity scores and ability to tolerate F. avenaceum infection without significant (p > 0.05) reductions in plant height, shoot dry weight, and root dry weight. Among the varieties with nonpigmented flowers and seed coats, only Cameor showed partial resistance to F. avenaceum when challenged with reduced conidial concentration. Root dry weight (R = −0.86), plant height (R = −0.82), and shoot dry weight (R = −0.78) had a strong negative correlation (p < 0.001) with disease severity, suggesting that F. avenaceum root rot can negatively impact the growth and development of pea seedlings. F. avenaceum resistance identified in this study can be utilized to study the molecular basis of the resistance and develop disease-resistant varieties. While our findings suggest a relationship between pigmentation and F. avenaceum resistance, future research with a larger, more diverse panel is warranted to validate these initial results.

Introduction

Pea (Pisum sativum L.) is a rich plant-based source of dietary protein, carbohydrates, micronutrients, fiber, vitamins, and minerals for humans and livestock (Felix et al. 2017). Garden peas have been highly utilized by various food industries, while field peas are popularly cultivated for human consumption and livestock feed (Arnoldi et al. 2015). The demand for peas in the food market as ingredients has increased substantially in recent years, due to the growing consumer interest in plant-based protein as food ingredients, beverages, and noodles as an alternative to animal-based protein (Jha et al. 2021; Warkentin et al. 2015). Consequently, global pea production has increased in the last decade with substantial contributions to the export market of many countries in the world. Following soybeans and dry beans, pea is ranked third in terms of world production of legumes (FAOSTAT 2022). Canada is the largest field pea producer in the world followed by the Russian Federation, China mainland, India, and the United States, with most of the production in Canada coming from the prairies (FAOSTAT 2022). However, the productivity of field pea is threatened by root rot caused by several fungal and oomycete pathogens that form the pea root rot complex (Chatterton et al. 2019; Jha et al. 2021; Sharma et al. 2022). This root rot complex includes Aphanomyces euteiches, Fusarium avenaceum, F. solani f.sp. pisi, F. graminearum, F. redolens, and F. acuminatum (Banniza et al. 2013; Chatterton, Bowness, and Harding 2015; Zitnick-Anderson, Simons, and Pasche 2018). Many of these pathogens are ubiquitous in pea-growing regions of the world and can cause significant yield loss (Banniza et al. 2013; Sharma et al. 2022; Willsey et al. 2018).

F. avenaceum is the most aggressive Fusarium species in the root rot complex of pea (Moparthi et al. 2021; Safarieskandari, Chatterton, and Hall 2021; Willsey et al. 2018). On the Canadian prairies, F. avenaceum was identified as the most dominant Fusarium species causing root rot in pea (Chatterton et al. 2019; Feng et al. 2010) and accounted for up to 80% of the pathogens isolated from diseased roots in commercial pea crops (Feng et al. 2010). F. avenaceum also causes diseases in other important crops on the Canadian prairies including wheat (Tyburski 2014) and canola (Chen et al. 2014). Similarly, F. avenaceum was also found to be highly aggressive on pulse crops including pea, lentil, and chickpea, as well as on barley and wheat in the western United States (Moparthi et al. 2021). The root rot complex negatively impacts plants by restricting the transport of nutrients and water and reducing canopy density and uniformity in crop maturity (Xi, Stephens, and Hwang 1995). To date, there is no single control measure for root rot in field pea, as cultural practices have not been highly effective (Jha et al. 2021; Sharma et al. 2022).

Host plant resistance is the single most realistic, cost-effective, and sustainable strategy for the control of pea root rot disease. Although there is no complete resistance to pathogens of the root rot complex, partial resistance under a quantitative mode of action can offer durable and reliable resistance to plant pathogens (Zhan et al. 2015). Sources of partial resistance to root rot pathogens, especially Aphanomyces and F. solani, have been identified in pea germplasm (Bodah et al. 2016; Davis et al. 1995; Desgroux et al. 2016; Grünwald, Coffman, and Kraft 2003; Malvick and Percich 1999). The discovery of these various sources of partial resistance has resulted in the detection of numerous quantitative trait loci (QTLs), potential candidate genes, which is slowly revealing the molecular mechanisms controlling resistance to A. euteiches and F. solani f.sp. pisi (Coyne et al. 2019; Hamon et al. 2013; Lavaud et al. 2016; Williamson-Benavides et al. 2020). Consequently, this information has been utilized to advance resistance breeding efforts against these pathogens. Li et al. (2012) identified a QTL contributing to F. avenaceum resistance in pea, indicating that this trait is quantitative and is regulated by more than one gene. However, no study has been conducted to evaluate diverse pea accessions for F. avenaceum resistance and provided options for selecting candidate lines with the best available resistance to develop mapping populations and identify genes for resistance breeding in pea against F. avenaceum.

Flower and seed coat pigmentation due to polyphenols, a large group of secondary metabolites, plays an important role in plant defense responses to various fungal pathogens (Jha et al. 2021; Karre et al. 2019; Lattanzio, Lattanzio, and Cardinali 2006; Mendu et al. 2022). Activation of flavonoid biosynthetic genes is required for pigment production in seed coats, flowers, and other plant tissues (Harker, Ellis, and Coen 1990; Mendu et al. 2022; Paulsmeyer and Juvik 2023). Previous studies have established that pea accessions with pigmented flowers and seed coats have higher levels of resistance to Fusarium root rot caused by F. solani f.sp. pisi, compared to accessions with nonpigmented flowers and seed coats (Bodah et al. 2016; Grünwald, Coffman, and Kraft 2003). Although a moderate level of resistance to root rot caused by F. solani f.sp. pisi and F. avenaceum was reported in few pea accessions with nonpigmented flowers (Bodah et al. 2016; Li et al. 2012; Wu et al. 2022), it is not clear how different pea accessions and varieties with pigmented or nonpigmented flowers will respond to a highly aggressive F. avenaceum isolate.

Therefore, the main objective of this research was to identify pea accessions and varieties of diverse global origin with pigmented or nonpigmented seed coats and flowers and from different market classes with moderate to high levels of resistance to F. avenaceum. Such lines would be prime parental lines for the introgression of F. avenaceum resistance into high-yielding pea cultivars in breeding programs. Additional objectives were to determine and compare the impact of root rot severity on growth indicators such as plant height, shoot dry weight, and root dry weight among the accessions and varieties and to assess correlations between root rot severity values and the growth indicators.

Materials and Methods Plant Material

Twenty pea accessions with either pigmented or nonpigmented flowers and seed coats representing different market classes were used in this study. These accessions originated from diverse regions of the world (Table 1 and Figure 1a). In addition to seed coat types and origins, these accessions were selected based on their agronomic characteristics (i.e., yield, flower type, and disease resistance to other pathogens) and wide use either as germplasm or cultivar. Before initiating experiments, germination rates for the accessions were confirmed as 80% or higher.

TABLE 1 Pea (Pisum sativum L.) accessions evaluated for Fusarium avenaceum root rot resistance.

Accession	Market class	Seed coat color	Origin	Source
Cameor	Yellow	Nonpigmented	France	Seminor
CDC Lewochko	Yellow	Nonpigmented	Canada	CDC USASK
No. 9292 (PI 195020)	Dun	Pigmented	Ethiopia	WRPIS
CDC Amarillo	Yellow	Nonpigmented	Canada	CDC USASK
CDC Dakota	Dun	Pigmented	Canada	CDC USASK
AAC Profit	Yellow	Nonpigmented	Canada	AAFC
CDC Acer	Maple	Pigmented	Canada	CDC USASK
CDC Blazer	Maple	Pigmented	Canada	CDC USASK
CDC Limerick	Green	Nonpigmented	Canada	CDC USASK
CDC Meadow	Yellow	Nonpigmented	Canada	CDC USASK
CDC Rocket	Maple	Pigmented	Canada	CDC USASK
CDC Vienna	Maple	Pigmented	Canada	CDC USASK
Champagne	Fodder	Pigmented	France	INRAE
Moravska krajova	Fodder	Pigmented	Czechia	Unknown
Morgan	Dun	Pigmented	Australia	WWAI
PBA OURA	Dun	Pigmented	Australia	PBA
PI 175231	Maple	Pigmented	Nepal	WRPIS
PI 280609	Maple	Pigmented	Former Soviet Union	WRPIS
PI 639977	Maple	Pigmented	Bulgaria	WRPIS
90-2079	Green	Nonpigmented	USA	WRPIS

Abbreviations: AAFC = Agriculture and Agri-Food Canada, Lacombe Research and Development Centre, Canada; CDC USASK = Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada; INRAE = The French National Research Institute for Agriculture, Food and the Environment, Paris France; PBA = Pulse Breeding Australia, Australia; Seminor = Seminor French Breeding Company; WRPIS = Western Regional Plant Introduction Station, Pullman, WA, USA; WWAI = Wagga Wagga Agriculture Institute, NSW Australia.

FIGURE 1. (a) Diverse pea accessions with pigmented and nonpigmented seed coats evaluated for resistance to Fusarium avenaceum root rot. (b) High-resolution microscopic observation of F. avenaceum at 40× magnification. (c) Front and reverse sides of F. avenaceum colony under incubation in potato dextrose agar plate. (d) Noninoculated pea plants with no visible symptoms and inoculated plants showing low to high and (e) very severe visible disease symptoms of F. avenaceum root rot.

F. avenaceum Isolates

Six F. avenaceum isolates (Fav01, Fav02, Fav03, Fav05, Fav14, and Fav15) from the pulse crop pathogen collection of the Crop Development Centre (University of Saskatchewan) previously isolated from symptomatic roots of pea, chickpea (Cicer arietinum), or lentil (Lens culinaris) collected in fields at Meath Park, Assiniboia, or at the North Seed Farm or in a root rot nursery at the University of Saskatchewan, Saskatchewan, were single-spored and identified as F. avenaceum based on their sequences of the partial elongation factor 1-α (TEF1-α). Briefly, pieces of washed root tissue (~1 cm) from the field with disease lesions were surface sterilized in 10% bleach for 2 min, rinsed with sterile distilled water (sdH₂O), placed on potato dextrose agar (PDA, Difco Potato Dextrose Agar, Becton, Dickinson and Co., MD, USA) plate, and incubated at 23°C–25°C under fluorescent light for 4 days. Four plugs were transferred from the colony growing out of the root tissue to a fresh PDA plate and incubated under the same condition. Five plugs from the new culture were transferred into sdH₂O and vortexed. After the suspension was serially diluted, 500 μL of each dilution was pipetted into sterile Petri dishes containing water agar and incubated for 24 h under light. A single spore of each of the isolates was gently picked under a stereomicroscope, placed on separate PDA plates, and incubated for 6 days at 23°C–25°C under fluorescent light (Zitnick-Anderson, Simons, and Pasche 2018). Agar plugs of the single-spored culture were placed in glycerol cryogenic stock (20% glycerol) for storage at −80°C. In a preliminary experiment, the isolates were screened for their pathogenicity using three pea varieties: CDC Dakota, CDC Amarillo, and CDC Meadow. Among the six isolates, Fav01, originally isolated from a pea root, was selected as the most aggressive isolate in our test (Figure S1 and Table S1) and was used for germplasm screening here.

Inoculum Preparation

Conidial suspension of Fav01 used in this study was produced by retrieving an agar plug from the cryogenic culture storage at −80°C, placing it on PDA amended with 1 mL/L chloramphenicol (AMRESCO, OH, USA) and incubating the culture for 6 days (Figure 1c). After incubation, three plugs from the growing edge of the colony were used to inoculate each 250-mL flask containing 100 mL of CMC (carboxymethyl cellulose sodium salt, TCI Co., Ltd., Tokyo, Japan) broth, previously shown to promote conidiation of F. avenaceum (Foroud et al. 2012; Safarieskandari, Chatterton, and Hall 2021), which were incubated for 5–6 days at 23.5°C on an incubator shaker at 150 rpm with the lights on. Conidia were spun down at 3700 rpm for 2 min, rinsed three times with sdH₂O followed by centrifugation, and strained through a double layer of cheesecloth to remove mycelia. The conidia concentrations were estimated using a hemocytometer (Figure 1b), and the suspension was diluted to the final concentrations of 7.5 × 10³ conidia mL⁻¹ water for inoculation. A fresh inoculum was produced for each repeat of the experiments.

Plant Growth Conditions

The seed soak method (Porter et al. 2015) with some modifications was used for the inoculation of the seeds. This method was preferred due to its efficiency, the reproducibility of results, and the simplicity of setup, as evident in our preliminary experiments. The seeds of each accession were surface sterilized in 70% ethanol for 30 s and rinsed with sdH₂O once. The seeds were then soaked in 10% bleach solution (NaOCl) amended with 2 drops of Tween 20 for 3 min and rinsed with sdH₂O three times. An equal number of sterilized seeds of each variety and accession was placed in 50-mL centrifuge tubes, and a standard volume of inoculum suspension or sdH₂O was poured over the seeds to submerge them. The sterilized seeds were soaked for 4 h in either Fav01 conidia suspension or in sdH₂O for the noninoculated control set, after which they were drained and planted immediately. Plastic pots (100 mm, Kord Products, ON, Canada) were filled with vermiculite (Holiday Vermiculite, Medium-Fine 4 cu ft, Montreal, CA) and moistened uniformly before seeding. Four seeds per accession were sown in a pot with four replicate pots per treatment (inoculated vs. noninoculated control). The pots were arranged in a split-plot design, with each accession randomly arranged within subplots and the treatment as the main plot. The plants were irrigated as needed. The experiment was conducted in a Conviron growth chamber (model: GR178) located at the College of Agriculture and Bioresources, University of Saskatchewan. Chamber conditions were maintained at day/night temperatures of 25°C/10°C with a 16-h photoperiod. The experiment was conducted twice under the same conditions.

To rule out the possibility of overwhelming the plant resistance responses from the inoculum concentration of 7.5 × 10³ conidia mL⁻¹ water, a follow-up experiment was conducted with lower inoculum concentrations. This was necessary for the detection of partial resistance to the highly aggressive Fav01 in nonpigmented flower pea varieties with the seed-soak inoculation method. In this experiment, the seven pea varieties with nonpigmented flowers were evaluated together with CDC Dakota as the partially resistant check. Inoculum suspensions were prepared as previously described and adjusted to 5 × 10³, 2 × 10³, and 5 × 10² conidia mL⁻¹. Each accession was separately treated with the three inoculum concentrations and sdH₂O with 4 replications per treatment. The experiment was set up as previously described and was conducted twice. The control plants of accession 90-2079 exhibited poor growth (40% emergence rate) in this follow-up trial and were excluded from our analysis.

Root rot severity, plant height, root dry weight, and shoot dry weight of inoculated and noninoculated control plants were recorded 21 days after planting to assess disease impacts on plant development. Recorded values for individual plants within a pot were averaged to represent one replicate. Root rot severity was assessed as described by Wang and Gottwald (2017) and Porter et al. (2015) with modifications based on hypocotyl discoloration using a visual scale from 0 to 10 (0 = no disease; 10 = plant died or failed to emerge). The extension scale ranges from: 0, no lesion; 1 = 5% root rot severity; 2 = 15%; 3 = 25%; 4 = 35%; 5 = 45%; 6 = 55%; 7 = 65%; 8 = 75%; 9 = 85%; 10 = 95%, plant died or failed to emerge (Figure 1d,e). The relative reduction (percentage change) in plant height, shoot dry weight, and root dry weight was calculated as the ratio between the trait expression of diseased plants and trait expression of control plants: [Image Omitted. See PDF]

Statistical Analysis

Data analyses were conducted using R software version 4.1.3 (R Core Development Team 2022). The homogeneity of variances of root rot severity, plant height, shoot dry weight, and root dry weight in the two replicated trials were assessed with Levene's test using the car package (Fox and Weisberg 2018). Repeats of the experiments had homogenous variances (p > 0.05); hence, the data were pooled for analysis. To test the effect of inoculum treatment and accession on each of the variables, an analysis of variance (ANOVA) was performed using mixed-effects models with each of the response variables. These models also included accession, treatment, and their interaction as factorial fixed effects, with both repeat and replication considered as random factors using the mixed model procedure (Kuznetsova, Brockhoff, and Christensen 2017). Mean separation (post hoc) analysis was performed using the estimated marginal means procedure (Russell 2018), and estimated marginal means are presented and discussed. To compare the relative impact of disease on trait expression among the accessions, a separate ANOVA was performed using mixed-effects models. The models included either percent height reduction, percent root dry weight reduction, or percent shoot dry weight reduction as the response variable with accession as the fixed effect, and both repeat and replication considered as random factors. Mean separation was performed as previously described. To test the association between root rot severity and plant height, shoot dry weight, and root dry weight, a Pearson correlation analysis was performed on the data from the inoculated treatment.

Disease Verification

Root tissues of infected plants from three inoculated plants were randomly taken from each test to verify the presence of F. avenaceum in infected tissue. The root tissue was surface sterilized and plated onto PDA as previously described. Culture growth and morphology were observed under a microscope and compared with the original cultures to confirm that the disease symptom was due to the F. avenaceum inoculation.

Results Root Disease Severity Caused by F. avenaceum

Mean root rot severity was significantly affected by accession, treatment, and their interaction (p < 0.001). Root rot severity was 98.8% lower in noninoculated than inoculated plants, and the difference was statistically significant for all accessions (p < 0.05). This confirmed that natural infection and cross-contamination were minimal and negligible in the seeds and during the experiment for all accessions. Root rot severity (8.0%–43.0%) in the pigmented flower accessions was significantly lower compared to the nonpigmented varieties (89.7%–95.0%) (Table 2). The highest levels of resistance to F. avenaceum were observed in CDC Acer, followed by CDC Vienna, PI 639977, Champagne, and PBA OURA, all of which are pigmented accessions and showed very little infection after inoculation (Table 2). Among the nonpigmented flower varieties, Cameor had a slightly lower root rot severity compared to other nonpigmented flower varieties; however, there were no significant differences, as they clustered into one group which differentiated them from the pigmented varieties (Table 2).

TABLE 2 Root rot severity of pea accessions with pigmented versus nonpigmented seed coats after inoculation with Fusarium avenaceum, ordered from the lowest to the highest scores, results averaged for 32 individual plants per accession from two tests.

Accession	Seed coat color	Mean root rot severity (%)a	Groupingb
CDC Acer	Pigmented	8.0	ABC
CDC Vienna	Pigmented	11.3	ABCD
PI 639977	Pigmented	11.4	ABCD
Champagne	Pigmented	12.2	ABCD
PBA OURA	Pigmented	14.1	ABCDE
Morgan	Pigmented	15.3	ABCDE
CDC Blazer	Pigmented	16.4	BCDE
CDC Dakota	Pigmented	20.4	CDE
PI 175231	Pigmented	23.1	CDE
PI 280609	Pigmented	23.6	DE
Moravska krajova	Pigmented	26.4	DE
CDC Rocket	Pigmented	29.2	EF
No. 9292	Pigmented	43.0	F
Cameor	Nonpigmented	89.7	G
90-2079	Nonpigmented	95.0	G
CDC Lewochko	Nonpigmented	95.0	G
AAC Profit	Nonpigmented	95.0	G
CDC Amarillo	Nonpigmented	95.0	G
CDC Limerick	Nonpigmented	95.0	G
CDC Meadow	Nonpigmented	95.0	G

^aEstimated marginal mean of root rot severity values at 21 days after inoculation based on a rating scale of 0 to 10 extended to 0 to 95% with a score of 95% being the most severe at inoculum concentration of 7.5 × 10³ conidia mL⁻¹.

^bMeans with the same letters are not significantly different from one another at p ≤ 0.05 based on Tukey adjustment.

We conducted a follow-up study to test whether the F. avenaceum inoculum concentration was possibly overwhelming the resistance responses of the nonpigmented flower varieties. In this study, mean root rot severity was also significantly affected by accession, treatment, and their interaction (p < 0.05; Figure 2). All accessions had significantly lower root rot in noninoculated control plants compared to the inoculated plants, except for the partially resistant pigmented check, CDC Dakota, for which scores for inoculated and noninoculated control plants were near zero and were not significantly different from each other (p < 0.05; Figure 2). Despite the reduced inoculum concentrations, all nonpigmented flower accessions were susceptible when inoculated with Fav01 conidia at concentrations of 5 × 10², 2 × 10³, and 5 × 10³ conidia mL⁻¹, except Cameor, which had an intermediate level of resistance with 69.7% and 49.8% root rot severity after inoculation with 5 × 10² and 2 × 10³ conidia mL⁻¹, respectively (Figure 2). Inoculation of Cameor at 2 × 10³ conidia mL⁻¹ resulted in similar root rot severity to that of CDC Dakota after inoculation with 5 × 10³ conidia mL⁻¹ (p = 0.365; Figure 2).

FIGURE 2. Effects of inoculum concentrations on Fusarium avenaceum root disease severity of pea accessions with nonpigmented seed coat and flower. Means with the same letters are not significantly different among the varieties and treatments at p ≤ 0.05. Error bars indicate the standard error of the mean. CDC Dakota was included as a pigmented-resistant check.

Impact of F. avenaceum on Plant Growth Indicators—Height, Root, and Shoot Dry Matter

F. avenaceum root rot had a significant negative correlation with plant height based on a correlation analysis using data from all accessions (p < 0.001; R = −0.82). However, plant height for the pigmented accessions CDC Acer, CDC Blazer, CDC Dakota, CDC Vienna, Champagne, Morgan, PBA OURA, and PI 280609 did not differ significantly between inoculated and noninoculated treatments (p > 0.05; Figure 3). For other accessions including all those of the nonpigmented flower type, inoculated plants were significantly shorter than noninoculated plants (p < 0.05; Figure 3). While there were no differences in height reduction due to Fav01 inoculation among nonpigmented flower accessions or most of the pigmented accessions except between CDC Dakota and PI 639977, the magnitude of reduction in plant height due to F. avenaceum infection was substantially greater in the accessions with nonpigmented flowers (91.6%–100%) compared to the pigmented accessions (−3.6% to 21.1%) (Figure 3).

FIGURE 3. Plant height reduction showing the effect of inoculation with Fusarium avenaceum on pea accessions 21 days after inoculation based on the mean difference in height (cm) between noninoculated and inoculated plants combined from two tests. Asterisks and ns indicate significant and nonsignificant differences, respectively, between inoculated and noninoculated controls based on simple degree contrast for treatment at p ≤ 0.05. Means with the same letters are not significantly different in the magnitude of height reduction among the accessions at p ≤ 0.05. Bars in darker and lighter shades of purple represent accessions with pigmented and white flowers, respectively.

Across all accessions, F. avenaceum root rot had a significant negative correlation with shoot dry weight (p < 0.001; R = −0.78). Shoot dry weight for the pigmented accessions CDC Acer, CDC Blazer, CDC Dakota, CDC Rocket, CDC Vienna, Champagne, Morgan, No. 9292, PBA OURA, and PI 280609 did not differ significantly between inoculated and noninoculated treatments (p > 0.05; Figure 4). Other pigmented accessions and all the nonpigmented accessions had significantly lower shoot dry weight in inoculated than in noninoculated treatments (p < 0.05; Figure 4). Additionally, the magnitude of reduction in shoot dry weight due to F. avenaceum infection was significantly greater in the nonpigmented accessions (94.5%–100%) than in the pigmented accessions (−18.9% to 31.6%), and no significant differences in shoot dry weight reduction were observed among the former (Figure 4). Similarly, among the pigmented flower accessions, no significant differences in shoot dry weight reduction were observed due to F. avenaceum infection, except between No. 9292 and Morasvska krajova, PI 175231, and PI 639977 (Figure 4).

FIGURE 4. Shoot dry weight reduction showing the effect of inoculation with Fusarium avenaceum on pea accessions 21 days after inoculation based on the mean difference in shoot dry weight (g) between noninoculated and inoculated plants combined from two tests. Asterisks and ns indicate significant and nonsignificant differences, respectively, between inoculated and noninoculated controls based on simple degree contrast for treatment at p ≤ 0.05. Means with the same letters are not significantly different in the magnitude of shoot dry weight reduction among the accessions at p ≤ 0.05. Bars in darker and lighter shades of purple represent accessions with pigmented and white flowers, respectively.

F. avenaceum root rot also had a significant negative correlation with root dry (p < 0.001; R = −0.86), and root dry weight differences between inoculated and noninoculated treatments for the pigmented accessions CDC Acer, CDC Blazer, CDC Dakota, CDC Rocket, CDC Vienna, Morasvska krajova, Morgan, No. 9292, PBA OURA, PI 175231, and PI 280609 did not differ significantly (p > 0.05; Figure 5). Other pigmented accessions and all the nonpigmented accessions had significantly less root dry weight in inoculated than in noninoculated treatments (p < 0.05; Figure 5). As before, the magnitude of reduction in root dry weight due to F. avenaceum infection was significantly greater for the nonpigmented flower accessions (96.8%–100%) than for the pigmented accessions (−14.6% to 28.1%). Root dry weight reduction due to F. avenaceum infection neither differed among the nonpigmented flower accessions nor the pigmented flower accessions, except between No. 9292 and Champagne, PI 280609, and PI 639977 (Figure 5).

FIGURE 5. Root dry weight reduction showing the effect of inoculation with Fusarium avenaceum on pea accessions 21 days after inoculation based on the mean difference in root dry weight (g) between noninoculated and inoculated plants combined from two tests. Asterisks and ns indicate significant and nonsignificant differences, respectively, between inoculated and noninoculated controls based on simple degree contrast for treatment at p ≤ 0.05. Means with the same letters are not significantly different in the magnitude of root dry weight reduction among the accessions at p ≤ 0.05. Bars in darker and lighter shades of purple represent accessions with pigmented and white flowers, respectively.

Discussion

This study evaluated resistance to F. avenaceum isolate Fav01 from Saskatchewan in 20 geographically diverse pea accessions with pigmented or nonpigmented flowers and seed coats representing different market classes. Identification of pea accessions with partial resistance and susceptibility to F. avenaceum and the genotype-dependent impact of this pathogen on plant growth and development provides an opportunity for selecting candidates for the development of a mapping population, QTL mapping, and resistance gene identification. This research utilized a highly aggressive F. avenaceum isolate from the Saskatchewan growing region, the leading producer of field peas in Canada (FAOSTAT 2022). Occurrence of root rot was previously observed in every pea field surveyed in the Canadian prairies where F. avenaceum was implicated as the most common and aggressive Fusarium species of the pea root rot complex (Chatterton et al. 2019). Based on root rot assessments and the ability to withstand F. avenaceum infection without any negative impact on growth and development, CDC Acer, CDC Vienna, PBA OURA, Morgan, CDC Blazer, CDC Dakota, and PI 280609 with pigmented flowers and seed coats were identified as the most resistant accessions (Table 2). Cameor, a white flower variety with nonpigmented seed coat, has a moderate level of F. avenaceum resistance (Figure 2). Given their good agronomic traits, many of these accessions could therefore be utilized in breeding programs within their respective market classes.

Our results with a Canadian isolate of F. avenaceum are consistent with findings of previous studies in the United States where a high level of partial resistance to F. solani f.sp. pisi infection was observed in pigmented flower genotypes compared with the nonpigmented genotypes (Bodah et al. 2016; Grünwald, Coffman, and Kraft 2003; Kraft 1975). In our study, root rot severity after F. avenaceum inoculation ranged from 8.0% to 43.0% in the pigmented flower accessions. In contrast, all the white-flowered accessions expressed highly susceptible reactions with scores of 89.7%–95.0%, accompanied by stunting and seedling death at the early growth stages. Severity levels of 95.0% due to F. avenaceum inoculation were observed in all white-flowered accessions except Cameor, which showed a numerically, but not significantly, lower root rot score of 89.7%. Given the high aggressiveness of Fav01 used in this current study, we reduced the initial inoculum concentration by ~30%, ~70%, and ~90%, to rule out the possibility of overwhelming the resistance responses of the white-flowered accessions beyond the threshold at which a moderate level of genetic resistance may be detected. Regardless of the inoculum concentration, all the white-flowered accessions expressed highly susceptible reactions except Cameor which had a significantly lower root rot severity indicative of a moderate level of partial resistance (49.8% RDS score) when the inoculum concentration was reduced by ~70%. This is consistent with previous studies that reported partial resistance to root rot caused by F. solani f.sp. pisi (Bodah et al. 2016; Grünwald, Coffman, and Kraft 2003) and F. avenaceum (Li et al. 2012; Wu et al. 2022) in some white flower pea accessions. Our results indicate that while the initial concentration may be appropriate for detecting a high level of partial resistance, a reduced concentration of conidia may be necessary when inoculating with a highly aggressive isolate to identify genotypes that may possess a moderate level of partial resistance to F. avenaceum.

The A-gene that codes for pigmented flowers and seed coats is responsible for higher partial resistance to F. solani f.sp. pisi in pea (Weeden and Porter 2007). Various transcription factors and enzymes are involved in the phenylpropanoid biosynthetic pathway, leading to pigmentation in various plant tissues (Welch, Wu, and Simon 2008). A time-course transcriptomic study conducted by Williamson-Benavides et al. (2020) showed that the F. solani f.sp. pisi-resistant pea genotypes had a higher level of expression of genes in the phenylpropanoid pathway such as flavonoid biosynthesis, flavone, and flavonol biosynthesis, under the basal conditions, compared to the susceptible genotypes. In the current study, we observed root rot scores of 2.3%–27.2% for CDC Dakota, a pigmented flower variety, and 82.2%–95.0% for CDC Amarillo, a white-flowered variety. Seed components of these two varieties and the recombinant inbred lines (PR-20) derived from their cross were previously characterized by our group for polyphenolic profile (Jha et al. 2019). The study reported that phenolic compounds such as flavan-3-ols, and flavonols were present in substantially greater concentrations (1300–6100 times) in seed coats of pigmented flower lines compared with white flower lines. This suggests that while pigmentation-related genes may additively enhance F. avenaceum resistance, they may not completely determine the resistance phenotype in peas as partial resistance in nonpigmented peas is shown here and in previous studies (Bodah et al. 2016; Li et al. 2012; Wu et al. 2022). Further, the accumulation of flavonoid intermediates found in nonpigmented seed coats (Jha et al. 2019) may explain the lower level of resistance to Fusarium root rot in white-flowered accessions relative to pigmented accessions. Although we could not rule out the possibility that the small sample size in our study could bias the relationship between pigmentation and F. avenaceum resistance, it seems likely that more individuals resistant to Fusarium root rot would be found in any pigmented population than in a nonpigmented population (Bodah et al. 2016; Grünwald, Coffman, and Kraft 2003) due to the antimicrobial properties of the anthocyanin and flavonoid pigments (Karre et al. 2019; Lattanzio, Lattanzio, and Cardinali 2006; Mendu et al. 2022). Future research with a larger, more diverse panel is warranted to validate these initial results and unravel the genetic linkage between pigmentation genes and resistance genes in response to F. avenaceum in peas.

Previous studies have assessed the impact of biotic stress on plants by estimating reductions in plant height, shoot dry weight, and/or root dry weight (Awodele and Bennett 2022; Bodah et al. 2016; Hannula et al. 2020). The current study demonstrated that the impact of F. avenaceum root rot on reductions in plant growth indicators (height, shoot dry weight, and root dry weight) depends on the variety rather than disease threshold values. These results are in contrast with the findings of Bodah et al. (2016) who reported that only the genotypes with root rot severity values of ≤ 3.05 (~50% of the rating scale) had no significant reductions in plant height, shoot dry weight, and root dry weight. In our study, however, Champagne and PI 639977 with the fourth and fifth lowest F. avenaceum root rot scores, respectively, experienced significant reductions in one or more of the growth predictors. This result is similar to the findings of Persson, Bødker, and Larsson-Wikström (1997) who reported a significant reduction in plant weights due to root rot in a pea variety with moderate disease symptoms. Further, root dry weight, shoot dry weight, and plant height all had a strong negative correlation with F. avenaceum root rot severity, indicating that F. avenaceum infection is closely associated with a reduction in shoot and root growth. This result is consistent with previous reports on the significant negative impact of Fusarium root rot on plant growth predictors in pea (Bodah et al. 2016) and wheat seedlings (Wang et al. 2015). Among the partial resistant accessions identified here, only CDC Acer, CDC Vienna, PBA OURA, Morgan, CDC Blazer, CDC Dakota, and PI 280609 did not exhibit significant reductions in all the growth predictors examined in response to F. avenaceum infection. This indicates that, in addition to limiting the amount of root damage, these varieties and accessions can also tolerate the presence of F. avenaceum without significant growth restriction. Our results suggest that selection of pea accessions based on their ability to resist the negative impact of F. avenaceum root rot on plant growth, rather than a disease threshold value, is a good criterion for breeders to select for partially resistant pea germplasm. Therefore, these pea varieties and accessions can be used as sources of high levels of partial resistance to F. avenaceum root rot for F. avenaceum resistance breeding of purple flower pea varieties.

The applicability of screening systems under controlled conditions to predict adult plant field resistance to Fusarium root rot has been established in previous studies (Porter et al. 2015; Wille et al. 2020). In the current research, the methodology used for testing the pea varieties and accessions for resistance to F. avenaceum root rot gave very repeatable results, as the results for the repeated trials were similar (p > 0.05). Our choice of inoculation method is consistent with previous studies where seed inoculation method was preferred and used for Fusarium spp. (Feng et al. 2010; Grünwald, Coffman, and Kraft 2003; Porter et al. 2015). However, the use of this method with a highly aggressive F. avenaceum isolate in this study, even at a relatively low inoculum concentration, resulted in highly susceptible reactions in some varieties. Seed inoculation may promote high levels of penetration of the developing pathogen into the host, which may facilitate rapid infection of germinating seedlings by Fusarium spp. in the susceptible varieties used in our study, consistent with the findings of Afshari, Hemmati, and Sheikh (2020). The high root rot scores observed in the current study may be reflective of the prevalent field conditions in the Canadian prairies, where moderate to severe root rot pressure occurs (Chatterton et al. 2019).

In conclusion, the evaluation of 20 pea accessions of diverse global origins has identified different sources of resistance to F. avenaceum root rot. These include pea accessions and varieties with high levels of resistance, moderate resistance, and susceptibility to this pathogen from different market classes and seed coat and flower color types. Further, a measure of root dry biomass, which was the plant growth predictor with the highest correlation with root rot severity, can be combined with a visual rating of disease symptoms to measure disease impact. Coselection for high dry root biomass and F. avenaceum root rot resistance trait may be beneficial for F. avenaceum resistance breeding in peas. The inoculation method used in this study can be used to assess different levels of resistance to F. avenaceum root rot in pea breeding programs and genetic studies. While this study presents its limitations due to the small sample size and lack of evidence of field resistance for the accessions, the germplasm, methods, and results reported in this study can provide direction for future studies. Future research with a larger, more diverse panel of accessions is required to validate these initial results under field and controlled conditions and to explore the possible association between F. avenaceum resistance and pigmentation genes.

Author Contributions

Stephen O. Awodele: Conceptualization; Formal analysis; Investigation; Methodology; Writing – original draft. Kishore K. Gali: Conceptualization; Investigation; Supervision; Writing – review and editing. Nimllash T. Sivachandra Kumar: Investigation; Writing – review and editing. Devini De Silva: Investigation; Writing – review and editing. Syama Chatterton: Conceptualization; Methodology; Supervision; Writing – review and editing. Sabine Banniza: Conceptualization; Methodology; Supervision; Writing – review and editing. Thomas D. Warkentin: Conceptualization; Project administration; Resources; Supervision; Writing – review and editing.

Acknowledgments

The authors acknowledge the technical support of Donna Lindsay and Brent Barlow at the Crop Development Centre, University of Saskatchewan. We would also like to thank the Saskatchewan Ministry of Agriculture and Saskatchewan Pulse Growers for their financial support.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data not directly published in this paper may be requested from the corresponding author.