Ascochyta blight, also referred to as the ascochyta blight complex, is one of the major diseases affecting field pea production and can be caused by several pathogens with anamorphs in the genus Ascochyta (Tivoli and Banniza, 2007). Worldwide, Peyronellaea pinodes (syn. Mycosphaerella pinodes), Ascochyta pisi, and Phoma pinodella have been associated with this disease. In Australia other species of Phoma including Phoma koolunga (Davidson et al., 2009), Phoma herbarum (Li et al., 2011), and Phoma glomerata (Tran et al., 2014) were also shown to be pathogenic on pea and have been associated with ascochyta blight. Among the causal agents of ascochyta blight, P. pinodes is considered most damaging with yield losses of 28–88% depending on environmental conditions (Bretag et al., 1995a; Tivoli et al., 1996; Xue et al., 1997; Garry et al., 1998). Symptoms of P. pinodes and Phoma pinodella are very similar with brown to purplish lesions of irregular shape and without a distinct margin (Jones, 1927). A. pisi, in contrast has light brown lesions with a distinct darker brown margin. Pycnidia are easily visible in mature lesions of A. pisi, but not in those of the other two species.

Infection of pea seed is one of the major survival mechanisms of Ascochyta spp. and an important way of transmission into previously uninfected areas, but for some species can also represent a major source of inoculum for the developing crop (Tivoli and Banniza, 2007). Infection reduces seed germination, and seedlings that do develop from infected seeds may be diseased resulting in poor plant development and stands (Jones, 1927; Maude, 1966; Moussart et al., 1998). Higher severity of seed staining could be correlated with deeper penetration of P. pinodes into the seed, which in turn reduced emergence rates (Moussart et al., 1998). Under controlled conditions, seed-to-seedling transmission was up to 100% for P. pinodes (Xue, 2000) and 40% for A. pisi (Maude, 1966).

The impact of seed-borne inoculum is influenced by factors including rainfall and temperature, and areas with low rainfall often produce disease-free seeds in the field (Bathgate et al., 1989; Bretag et al., 1995b). Surface sterilization of pea seeds results in a reduction of seed infection with P. pinodes by 60%, indicating that the pathogen may be mostly carried on the seed coat (Bathgate et al., 1989). Seed infection levels with P. pinodes higher than 10% can cause severe economic damage to the crop under conducive environmental conditions (Xue, 2000). Seed-borne infection of other species of the ascochyta blight complex such as Phoma spp. has not been identified as very important in initiating epidemics of ascochyta blight in the field. Ascochyta spp. can survive on pea seed coats for several years (Bretag et al., 1995b), and for A. pisi specifically, it was estimated that the fungus will be eliminated from seed after 5 to 7 years of seed storage in cool and dry conditions (Wallen, 1955).

Until 1961, A. pisi was the dominant pathogen recovered from pea seeds in Canada (Wallen et al., 1967a). Incidences of 85% seed infection with A. pisi, 27.5% with P. pinodes and 10% with Phoma pinodella were reported from Canada in the mid-1950s (Skolko et al., 1954). In 1961, the pea variety Century (originally released as Creamette [Gfeller and Wallen, 1961]) was introduced and quickly gained in acreage due to its high level of resistance to A. pisi. Simultaneously, P. pinodes became the dominant foliar pea pathogen in Canada (Wallen et al., 1967a). In the early 2000s, a resurgence of A. pisi was noted in western Canada based on increasing levels of this pathogen on harvested seeds (Morrall et al., 2011). In response to this, experiments were conducted to reassess the impact of seed infection in the epidemiology of A. pisi, to evaluate the importance of seed-to-seedling transmission under field conditions, and to determine the nature of seed-borne infection by A. pisi. It was hypothesized that pea plants developing from seeds infected with A. pisi would be infected with the pathogen and that seed infection would thus promote the development of A. pisi infection in the developing crop canopy. It was also hypothesized that low levels of seed coat staining would be indicative of no or a low incidence of embryo infection with A. pisi whereas high seed coat staining would be correlated with a high incidence of embryo infection.

Pea seedling emergence was slightly, but statistically significantly affected by the incidence of A. pisi infection of seeds. Based on the regression model here, an increase in the incidence by 7% A. pisi infection in seeds is predicted to reduce seedling emergence by 1 plant m^-1 representing 4% in our experiment with 26 plants m^-1. This indicates that even an incidence of 14.5% of seed infection, the highest infection level assessed here, will only have a minor impact on plant stands. A much more significant impact of A. pisi seed infection on emergence was reported previously by Jones (1927) who found 69 and 76% seedling emergence under field, and 75% under greenhouse conditions from a seed sample with an incidence of A. pisi infection of 8%, when compared to emergence of seeds from the same sample treated with organic mercuric dust. In contrast, assessments of seed samples from several years and locations with A. pisi infection rates of 10% resulted in seedling emergence of 85% (Wallen, 1955). In that study, samples with 44 and 46% A. pisi infections were assessed as well and had emergence rates of 87 and 67%, respectively, supporting observations here that A. pisi infection does not have a major impact on emergence, although the confounding impact of organisms other than A. pisi, observed in all of these studies, has not been quantified. When comparing these numbers it is important to keep in mind that the earlier reports used pea varieties that are now 60 to more than 100 years old, and were most likely more susceptible to A. pisi than modern CDC Patrick. Even though the first highly A. pisi resistant pea variety was only released in 1961 (Gfeller and Wallen, 1961), it can be speculated that pea varieties studied by Wallen (1955) may have already had improved resistance compared to those used by Jones (1927) 28 years earlier, as resistance to A. pisi will have been a primary breeding objective. A negligible impact of A. pisi on pea seedling emergence observed here is in stark contrast to P. pinodes where seed infection levels of 24 to 46% resulted in germination rates of 19 to 23% (Xue, 2000), and seeds with more than 50% seed coat staining had a seed-to-seedling transmission of 100% (Moussart et al., 1998).

Precipitation during the growing seasons of 2012 to 2014 at experimental locations was average or above average, so conditions generally were conducive for the initiation of epidemics. Very low levels of seedling infections and no effect of A. pisi seed infection on disease severity on the developing plants here indicated that infection of seeds with A. pisi used for seeding does not pose a risk for initiating epidemics in the field under western Canadian growing conditions. There was also no effect on seed yield, seed size or the infection levels with this pathogen of harvested seeds. In general, A. pisi is considered to be less aggressive than other pathogens, with reported yield losses of 11% compared to 45 and 25% due to P. pinodes and Phoma m. var pinodella, respectively (Wallen, 1965).

In four of the six field experiments seed infection of harvested seeds with A. pisi was higher than with P. pinodes despite the fact that for three of those four experiments, P. pinodes severity on pea plants was higher than A. pisi severity. Wallen et al. (1967b) pointed out a natural antagonism between A. pisi and P. pinodes, and also found that seed-borne infection tends to be higher with A. pisi compared to P. pinodes (Wallen, 1965). A higher incidence of A. pisi infection had been observed for certain seed lots in commercial seed testing labs in recent history as well (Morrall et al., 2011), which had triggered a re-assessment of the importance of A. pisi here. When assessing seed components for infection, the embryo of all seeds were infected with A. pisi irrespective of the amount of seed staining as long as there was some seed coat staining. This is distinctly different from seed infection by P. pinodes where the amount of seed coat staining is positively correlated with the depth of infection into the seed and the frequency of embryo infection (Moussart et al., 1998). For this pathogen, no necrosis on seed components other than on the seed coat was observed for seeds with less than 25% seed staining. Seeds with higher seed coat staining always showed evidence of necrosis caused by P. pinodes on the outward facing side of cotyledons, and a gradual increase in the incidence of necrosis on the inward-facing side of cotyledons from 12 to 100% as outer seed coat staining increased from 25 to 100%. Similarly, the incidence of necrosis on embryos increased from 10 to 100% once seed coat staining exceeded 25% and increased to 75 to 100%. This positive correlation between increasing outer seed coat staining and infection of inner seed components suggests that P. pinodes infects the more or less immature pod and penetrates from there into the seeds. The relatively high incidence of A. pisi in embryos and cotyledons irrespective of the amount of seed coat staining may indicate that A. pisi infection already occurs during flowering. The lack of correlation between foliar infection, from which water-splashed conidia could infect flowers, and the incidence or depth of seed infection indicates that airborne ascospores of A. pisi rather than water-splashed conidia may infect flowers and seeds, considering that windborne ascospores can be blown in from remote inoculum sources, and ascospore concentration will likely be more equal across a field. Little is known about the life cycle of A. pisi whereas that of P. pinodes has been well studied. The latter is homothallic and readily produces sexual fruiting structures (pseudothecia) which are thought to overwinter on pea stubble generating airborne ascospores that represent the initial inoculum for the new pea crop in the following season (reviewed in Roger and Tivoli, 1996). Studies in France showed that ascospores of P. pinodes are released throughout the growing season, but peak toward its end when large numbers of pseudothecia develop almost exclusively on senescent plant tissue, and mostly on stems of the maturing, increasingly diseased and senescent pea plants.

The teleomorph of the heterothallic species A. pisi, Didymella pisi, was only described relatively recently and it was shown that pseudothecia matured within 2 months at a constant temperature of 10°C, but their development ceased at 23°C (Chilvers et al., 2009). Historically, the daily maximum temperature in many parts of the Canadian Prairies exceeds 23°C during the growing season, but the daily average temperature often does not due to cool nights, so depending on the effect of fluctuating temperatures on perithecial development in A. pisi the climate may be conducive for ascospore production. To date, no studies have been conducted to determine whether pseudothecia develop under field conditions, nor have there been attempts to trap ascospores of this species. Indeed, such research would be complicated by the fact that P. pinodes tends to also be present. Although pseudothecia of A. pisi are slightly larger than those of P. pinodes, ascospores overlap in size (Punithalingam and Holliday, 1972, Chilvers et al., 2009), hence differentiating sexual structures of both species by microscopying or spore trapping would be highly complicated. Molecular probes readily differentiate between them, but do not allow to determine whether fruiting structures and spores are of sexual or asexual origin. In future, it may be possible to conduct studies of this nature through a combination of sophisticated imaging technology and molecular techniques.

Jones (1927) suggested that A. pisi overwinters as mycelium on pea straw after inoculating pea stems with this pathogen and incubating them under natural winter conditions in Wisconsin, United States. However, based on Wallen et al. (1967b) isolation of A. pisi from agricultural soil of eastern Canadian fields or from sterilized soil inoculated with spore suspensions of the ascochyta blight pathogens was unsuccessful whereas P. pinodes and Phoma pinodella were isolated on a regular basis. Incubation studies in sterilized soil each inoculated with one of the three ascochyta blight pathogens and incubated at temperatures ranging from -20 to +30°C revealed that A. pisi only survived in the soil for a period of 12 months at +5 and -20°C (Wallen and Jeun, 1968). At -20°C, P. pinodes and Phoma pinodella survived for that period as well, but with lower recovery rates than A. pisi. Both, P. pinodes and Phoma pinodella also survived up to 12 months in soil incubated at 5 to 25°C and were recovered at high rates, indicating clear temperature optima for A. pisi, and P. pinodes and Phoma pinodella. When sterilized soil was co-inoculated with the ascochyta blight pathogens in all possible pairwise combinations, P. pinodes was always recovered at the highest rate. In the presence of P. pinodes, Phoma pinodella survived for at least 9 months, whereas A. pisi was least competitive in the presence of either partner.

Jones (1927) also noted that seedlings developing from infected seed had lesions on the first leaves, so may represent a second source of initial inoculum. Testing commercial seed samples, Maude (1966) only found 40% of seed-to-seedling transmission for A. pisi compared to close to 100% for P. pinodes, and research here with a modern cultivar of pea revealed rare seed-to-seedling transmission under western Canadian field conditions. Considering that the pathogen is not readily isolated from soil (Wallen et al., 1967b), competes poorly with Phoma pinodella and P. pinodes in soil and does not, or rarely, produces chlamydospores (Wallen and Jeun, 1968), it can be speculated that infected seeds may play a much more important role for the survival of A. pisi than is the case for the other two common ascochyta blight pathogens. This would explain why the incidence of seed infection with A. pisi historically, and in some years in recent times, has been higher compared to P. pinodes.

The effect of A. pisi infection in seed on emergence was minimal under western Canadian growing conditions, A. pisi symptoms on seedlings were rare, and incidence levels of A. pisi infection of seed up to 14.5% did not increase the amount of disease on mature plants or harvested seeds. Infection with A. pisi of harvested seeds was common across all seed infection categories used for seeding, and staining was significant, so while seed infection up to the incidence level tested here may not impact pea production when the seed is used for seeding, the staining caused by A. pisi infection of seeds can result in lower quality of seeds to be sold as food or feed. The common infection of embryos and cotyledons of seeds of all staining categories may be indicative for a more dominant role of the seeds in the survival of A. pisi compared to P. pinodes that survives well in soil. Whether seed infection is initiated by ascospores during flowering, as speculated here, will only be revealed when more is known about the life cycle of this pathogen.

NSK conducted the research experiments as part of his MSc thesis, he contributed to the data analyses and to drafting the manuscript. SB was the principal investigator of this research project, supervised NSK, contributed to data analyses and the drafting of the manuscript.

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.