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Published online 2 October 2006
Published in Crop Sci 46:2409-2414 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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PLANT GENETIC RESOURCES

Genetic Resistance to Mycosphaerella pinodes in 558 Field Pea Accessions

Roger Zhanga, Sheau-Fang Hwangb,*, Kan-Fa Changc, Bruce D. Gossend, Stephen E. Strelkove, George D. Turnbulle and Stanford F. Bladec

a Alberta Research Council, Vegreville, AB T9C 1T4, Canada
b Alberta Agriculture, Food and Rural Development, Crop Diversification Centre North, Edmonton, AB T5Y 6H3
c Alberta Agriculture, Food and Rural Development, Field Crop Development Centre, Lacombe, AB T4L 1W1, Canada
d Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, SK S7N 0X2, Canada
e University of Alberta, Dep. of Agricultural, Food and Nutritional Science, Edmonton, AB T6G 2P5, Canada

* Corresponding author (Sheau-Fang.Hwang{at}gov.ab.ca)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Comprehensive assessments of germplasm collections of field pea (Pisum sativum L.) have failed to identify any accessions that are highly resistant to Mycosphaerella blight, caused by Mycosphaerella pinodes. In the present study, the broad-sense heritability of resistance to M. pinodes was studied on 558 pea genotypes in a detached-leaf assay and in field trials over 2 yr. In addition, analysis of covariance in disease reaction between the two assessment methods was examined. Only a few genotypes displayed relatively high levels of resistance, and as expected, no complete resistance was observed. The pattern of differences in reaction to M. pinodes among the genotypes demonstrates that resistance is quantitative and moderately heritable. Broad-sense heritability was higher in the detached-leaf assays than in the field trials, likely because of more environmental variance in the field. Covariance between repetitions of the detached leaf assays was not significant but was significant between the field tests. This indicates that detached-leaf assessments are more reliable for assessment of large numbers of accessions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FIELD PEA production in western Canada has increased steadily since the early 1980s, but Mycosphaerella blight can substantially reduce yields in the region (Chang et al., 1999). The principal cause of Mycosphaerella blight is Mycosphaerella pinodes (Berk. and Blox.) Vesterg. (anamorph: Ascochyta pinodes L.K. Jones), although Ascochyta pisi Lib., and Phoma medicaginis Malbr. & Roun. in Roun. var. pinodella (L.K. Jones) Boerema (syn. Ascochyta pinodella) can also be involved (Bretag and Ramsey, 2001). Genetic resistance would be the optimal approach to managing this disease (Wallen, 1965), but only a few genotypes with partial resistance have been identified (Marshall, 1962; Béasse et al., 1999). Breeding efforts have been hampered by a lack of highly resistant genomic resources, an incomplete understanding of the inheritance of resistance (Timmerman-Vaughan et al., 2002), and the absence of agreed-on methodologies for assessing resistance (McDonald, 1997).

A wide range of accessions have been evaluated for resistance to M. pinodes in western Canada (Xue et al., 1996) and in other production areas (Kraft et al., 1998; Fondevilla et al., 2005). Under field conditions, the reaction of some of these partially resistant pea lines has been inconsistent (Wallen, 1974; Wang et al., 2000). Variation in levels of resistance may be related to differences in the virulence of pathogen populations (Xue et al., 1998), regional and yearly variations in weather (Roger et al., 1999), and the different evaluation methodologies employed.

In the present study, 558 accessions of field pea were assessed for resistance to Mycosphaerella blight in two field trials, and the results were compared with those of detached-leaf assessments conducted under controlled conditions. Assessments under controlled environmental conditions were expected to reduce the effects of environmental factors and thereby improve assessment efficiency.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Pea Accessions and Inoculum
All 558 genotypes assessed in the study were obtained from the Western Regional Plant Introduction Station, USDA, Pullman, WA, USA. Four highly aggressive isolates of M. pinodes (Iso-1, Iso-2, Iso-3, and Iso-4) were collected from infected pea plants in Alberta, Canada, in 2003 (Hwang et al., 2006). Isolates were initially isolated as single hyphal tips and were then maintained on potato dextrose agar medium. For production of pycniospores, the isolates were grown on oat-water agar (OWA) medium [1.5% (w/v) agar, 1% (w/v) oat powder] for 4 wk at 20/15°C with a 16-h photoperiod at a light intensity of 300 µmol m–2 s–1. The OWA plates were flooded with sterile water and then scraped to release the spores. The spore suspensions were filtered through cotton gauze, the concentration of conidia was measured with a hemacytometer, and then adjusted to 100 spores µL–1 with sterile distilled water.

In the field experiments, the inoculum medium consisted of 5 kg peat moss, 1 kg autoclaved soil, and 0.5 kg sand. A fresh spore suspension (equal volumes of each isolate) was prepared for each inoculation, and 1 L of the suspension (100 spores µL–1) was applied to the medium. The inoculum was distributed evenly in the field plots at 1 x 107 spores m–2 at the early flowering stage (20–30 June).

Detached-Leaf Assay
Plant samples were collected from the field plots during vegetative growth, when most of the accessions had the seventh leaf completely extended. They were placed in plastic bags containing 50 mL of sterile water in a cooler at –1 to 5°C and transported to the laboratory. Plastic Petri dishes (10-cm diam.) were used as incubation chambers for a detached-leaf assay (Hwang et al., 2006). Briefly, the bottom of each dish was lined with an autoclaved paper towel, moistened with sterile water, and covered with a plastic mesh. The youngest fully expanded compound leaflet with a petiole (or a stipule for those lines without leaflets) attached to a short segment of stem was removed from each plant. The end of the petiole or stem was inserted through the mesh to absorb water from the paper towel. A pair of pea leaflets or a single stipule was tested in each dish. The leaflets or stipules were inoculated with 2 µL of fresh spore suspension containing 105 pycniospores mL–1 at each of four sites (one for each isolate). The leaflets and stipules were incubated at 22 ± 1°C and the lesions were measured after 5 d. Since the lesions often had an oval shape, both length and width were measured, and the area was calculated as follows:

Formula 1[1]
Disease severity was rated on a 0-to-4 scale, where 0 = no lesion development; 1 = lesion area of 1–33 mm2; 2 = 34 to 66 mm2; 3 = 67 to 100 mm2; 4 > 100 mm2. Since no significant differences were detected among the accessions for the four isolates, the blight severity data were pooled for analysis.

Field Trials
Field experiments were conducted at the Alberta Agricultural Field Crop Development Centre, Lacombe, AB, Canada, in 2003 and the research farm of the Alberta Research Council, Vegreville, AB, in 2004. Field pea had never been grown on either field. The trial was arranged in a randomized complete block design with three replications. Each plot consisted of two rows, 10 m in length and 30 cm apart, with plants spaced an average of 12 cm apart within the rows. Plots of the relatively susceptible line PI 179449 and the relatively resistant cv. Radley (Hwang et al., 2006) were planted every 40 rows as controls. The seed was planted in mid-May using a cone planter. Each genotype was rated for Mycosphaerella blight in mid-August at the late flowering stage, using a 0-to-9 scale (Xue et al., 1996).

Data Analysis
Analysis of variance for each trial was performed by PROC GLM, and variance components were calculated by PROC VARCOMP of SAS (SAS Institute Inc., Cary, NC). Restricted maximum likelihood estimates of the covariance components and correlation coefficients were obtained using PROC MIXED of SAS.

Analysis of covariance of field trials and detached-leaf assays across locations (Neter and Wasserman, 1974; Giauffret et al., 2000) was calculated as:

Formula 2[1]
where Yijk is the mean of blight severity for the ith accession, in the kth replicate of the jth location; µ is the grand mean; {alpha}i is the mean deviation for the ith accession; ßj is the mean deviation for the jth location; {theta}ij is the interaction term for the combination of the ith accession with the jth location; {delta}jk is the mean deviation for the kth replicate of the jth location; {varepsilon}ijk is the residual. It is assumed that the {alpha}i values sum to zero and thus have a population variance defined as:

Formula 3[3]
where a = 2 in our two situations. It is further assumed that ßj and {varepsilon}ijk are mutually independent and normally distributed with zero means and variances {sigma}2B and {sigma}2W, respectively. Thus, {sigma}2B and {sigma}2W represent the inter-individual (genotypic) and intra-individual (environmental) variance components (Symanski et al., 2000; Snedecor and Cochran, 1980). It follows that:

Formula 4[4]

Formula 5[5]
and

Formula 6[6]
Broad-sense heritability (H2) was estimated from variance components (Nyquist, 1991) as:

Formula 7[2]
where {sigma}2g is the genotypic variance; {sigma}2e is the error variance; {sigma}2gl is the genotype x location variance; r is the number of replications; and l is the number of locations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Variance estimates and the expected mean squares for each trial are listed in Table 1. In both the detached-leaf assays and field trials, the main effect of genotype was by far the largest source of variance. This indicated that the accessions differed substantially in disease reaction. The genotype x location interaction was significant in both the field trials and the detached-leaf assays.


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  		Table 1. ANOVA of the reaction of accessions of Pisum sativum to Mycosphaerella pinodes in field trials at Vegreville, AB, in 2003 and Lacombe, AB, in 2004.

 
A few of the accessions displayed relatively high levels of resistance, but complete resistance was not identified (Table 2). In Table 2, we list the 50 most resistant and 50 most susceptible accessions from the 558 genotypes assessed (based on mean rating across all tests), as we believe that both of these groups may be of interest to researchers working in this area.


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  		Table 2. Mean reactions of field pea accessions inoculated with Mycosphaerella pinodes as assessed in field trials (Field) and in a detached-leaf assay (Leaf) over 2 yr. The most resistant and most susceptible accessions from the 558 accessions tested are shown{dagger}.

 
The 558 accessions showed a continuous range (from low to high) of blight severity in the detached-leaf assays and field tests (Fig. 1). There was a strong positive correlation between the mean ratings across years for the detached-leaf assays and field trials (r = 0.96; P ≤ 0.001). However, broad-sense heritability in the detached-leaf assay was considerably higher than in the field trials (H2 = 0.85 vs. 0.55). The pattern of variance among the 558 lines indicated that inheritance of resistance to M. pinodes is quantitative (Fig. 2).


Figure 1
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  		Fig. 1. Disease reaction of 558 accessions of Pisum sativum to Mycosphaerella pinodes, based on the mean result over 2 yr of detached-leaf (A) assays and field trials (B). Means for the detached-leaf assay (A) were based on a 0-to-4 scale, and for field trials (B) on a 0-to-9 scale.

 

Figure 2
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  		Fig. 2. Blight severity in 558 accessions of Pisum sativum inoculated with Mycosphaerella pinodes in detached-leaf assays (A) and field trials (B) in 2003 and 2004. Severity was assessed 5 d post-inoculation in the leaf assays, and in mid-August in the field trials.

 
Covariance between years for the leaf assays was very low ({sigma}2B = –0.05) (Dudley and Moll, 1969) and not significant, indicating that the detached-leaf assay provided a consistent rating technique for analysis of large numbers of lines or accessions. Disease development between years was very similar, but the patterns do not overlap completely (Fig. 1-A).

Covariance of the detached-leaf assays with the field tests was highly significant (P ≤ 0.001) in 2003 ({sigma}2B = 1.04) and 2004 ({sigma}2B = 0.92), perhaps because of a high level of variance contributed by environmental factors in the field trials. Covariance between years of the field tests ({sigma}2B = 2.96) was highly significant (P ≤ 0.001), indicating that factors such as weather, presence of other diseases, and differences in pathogen population might have affected the expression and interpretation of resistance. However, linear trend lines representing disease severities of the population were very similar among genotypes at both locations (Fig. 1-B, linear 2003 and 2004), indicating the resistance of the population is stable and inherited.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The majority of the variance in disease reaction of accessions of P. sativum to M. pinodes resulted from genotype effects in both the detached-leaf assays and field trials. Resistance was expressed as a quantitative trait. However, expression of resistance was affected by the environment in the field trials. Resistance to Mycosphaerella blight was moderately heritable, with broad-sense heritability as high as 0.85 in the detached-leaf assays. Our results are consistent with previous reports indicating that small but heritable differences in susceptibility to Mycosphaerella blight exist among accessions and cultivars of P. sativum (Bretag, 1989; Fondevilla et al., 2005). As expected, no completely resistant accessions were identified.

A recent study (Hwang et al., 2006) showed that the disease reaction of field pea lines to M. pinodes in detached-leaf assays and field trials showed only a weak correlation for lines with intermediate levels of susceptibility. In contrast, data from the most susceptible and resistant lines were quite closely correlated in the detached-leaf assays and field trials. The lack of a strong correlation in disease reaction between the two assessments for many of the lines in the previous study (Hwang et al., 2006) led us to select analysis of covariance for use in this study. Analysis of covariance describes the association between two variables by combining analysis of variance and regression (Snedecor and Cochran, 1980). Because P. sativum is an inbred and diploid species, each accession was presumed to be a homogeneous (nonsegregating) population with a consistent reaction to M. pinodes, so variances within each genotype were the result of variances in environment, method, etc. Thus, variance due to heterogeneity (Neter and Wasserman, 1974), or variance of genotypes associated with more than one factor (genetic and environmental) (Giauffret et al., 2000; Rausher, 1992) can be detected in analysis of covariance with reduced errors.

Our results support the conclusion that it is possible to use detached-leaf assessments to reduce the number of lines that need to be assessed in the field. In our study, the correlation between the leaf and field assays was strong (r > 0.90). This indicated that when levels of disease are high in the field and occur in a larger population consisting of varied genotypes, the resistance ranking of lines corresponds very closely to their reaction in the leaf assays.

Regardless of the exact virulence mechanisms of the pathogen, previous reports have suggested that resistance to M. pinodes is often nonspecific (Wroth, 1998a), polygenically inherited, and quantitatively expressed against all genotypes of the pathogen (Parlevliet and Zadoks, 1977; Wroth, 1999). Resistance controlled by a major gene usually involves a specific elicitor that induces a defense reaction in the host (Bonas and Lahaye, 2002). However, M. pinodes secretes suppressors that inhibit the expression of resistance genes by the host (Shiriashi et al., 1992). As a result, the defense reaction of P. sativum is negatively regulated and is not activated when challenged by M. pinodes (Kiba et al., 1997; Dann and Deverall, 2000). Our results support these previous reports, confirming that resistance to M. pinodes is likely quantitative and controlled by multiple genes.

Limited knowledge of the genetic diversity of M. pinodes populations and the mechanism(s) of resistance in pea represent important obstacles in breeding for resistance to Mycosphaerella blight (Onfroy et al., 1999; Béasse et al., 2000; Bretag and Ramsey, 2001). Our results support previous conclusions that the pathosystem of M. pinodes on pea is complex (Clulow et al., 1991), and that further research is required to identify and elucidate the mechanism(s) of virulence and resistance (Fatehi et al., 2003).

Our results indicate that resistance to M. pinodes is sufficiently heritable to merit continued efforts to develop cultivars with partial resistance to this disease using traditional breeding methods (Wroth, 1998b; Darby et al., 1986). Pea genotypes with superior resistance to M. pinodes will be useful in breeding programs, in addition to their value in studies of host–pathogen interaction. However, identification and incorporation of genes for resistance from outside of P. sativum may be required for the development of highly resistant lines (Durieu and Ochatt, 2000).


   ACKNOWLEDGMENTS
 
We thank the Alberta Agricultural Research Institute, Alberta Crop Industry Development Fund, Western Grain Research Foundation, and Alberta Pulse Growers Commission for partial funding of the study, M. Hiltz for assistance with SAS, and C. Hundeby for technical assistance.

Received for publication February 9, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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