Lentil anthracnose: epidemiology, fungicide decision support system, resistance and pathogen races

Abstract

Colletotrichum lentis causes anthracnose of lentil (Lens culinaris) in Canada that results in defoliation, stem girdling and severe yield losses, but the disease is rarely reported elsewhere. The pathogen survives as microsclerotia on lentil debris for up to 3 years when buried in the soil, but loses viability on the soil surface. Windborne debris spreads the pathogen to neighbouring fields, while seedborne infection is less important. Foliar fungicides were registered, and a fungicide decision support system was developed which assessed disease risk with 85% accuracy. Around 2300 L. culinaris accessions from 50 countries were screened for resistance. Congruently, two races – Ct1 and Ct0 – were identified on differential lentil lines. Resistant lines were generated by cycles of inoculation and selfing of single resistant plants which resulted in the following three accessions resistant to both races: VIR2633 (Georgia), VIR2058 and VIR2076 (Czech Republic), while six and 49 lines had resistance to Ct0 and Ct1, respectively. Ct1 resistance is controlled by recessive and dominant genes crt1 and CtR3 in variety ‘Indianhead’, ctr2 and CtR5 in accession PI345629 and CtR4 in PI320937. Molecular markers linked to Ct1 resistance were identified on linkage group six, close to Ascochyta lentis resistance, and were used to combine resistance to both pathogens in breeding lines. Two repeat rich regions in the intergenic spacer (IGS) of ribosomal DNA can be used to differentiate the two C. lentis races. Utilizing length polymorphisms in a 39 nucleotide repeat region showed the races were equally frequent among isolates collected between 1991 and 1999, while 95% belonged to race Ct0 in 2010, likely because lentil varieties are susceptible to race Ct0, but around one-third of the varieties had Ct1 resistance.

Résumé

Colletotrichum truncatum cause l’anthracnose chez la lentille (Lens culinaris) au Canada, ce qui engendre la défoliation, l’annélation des tiges et d’importantes pertes de rendement, mais l’incidence de la maladie est rarement rapportée ailleurs. L’agent pathogène survit sous forme de microsclérotes sur les débris de lentilles, et ce, jusqu’à trois ans lorsqu’ils sont enfouis, mais perd sa viabilité lorsqu’ils sont laissés en surface. Les débris portés par le vent disséminent l’agent pathogène dans les champs voisins, tandis que l’infection portée par les semences est de moindre gravité. Des fongicides foliaires ont été homologués et un système d’aide à la décision relatif à leur application a été développé et permet d’évaluer le risque de maladie avec une précision de 85%. Environ 2300 accessions de L. culinaris provenant de 50 pays ont été criblées pour la résistance. Adéquatement, deux races, Ct1 et Ct0, ont été identifiées sur des lignées différentielles de lentilles. Des lignées résistantes ont été engendrées par des cycles d’inoculation et l’autofécondation de plantes résistantes individuelles, ce qui a produit les trois accessions suivantes résistantes aux deux races: VIR2633 (Géorgie), VIR2058 et VIR2076 (République tchèque), tandis que 6 lignées étaient résistantes à Ct0 et 49, à Ct1. Dans la variété ‘Indianhead’, la résistance à Ct1 est régulée par les gènes récessifs et dominants crt1 et CtR3, par les gènes ctr2 et CtR5 dans l’accession PI345629 et par le gène CtR4 dans PI320937. Les marqueurs moléculaires associés à la résistance à Ct1 ont été identifiés sur le groupe de liaison six, près de la résistance d’Ascochyta lentis, et ont été utilisés pour combiner la résistance aux deux agents pathogènes dans les lignées de sélection. Deux régions répétées riches de l’espaceur intergénique de l’ADN ribosomique peuvent être utilisées pour différencier les deux races de C. lentis. L’utilisation des polymorphismes de longueur dans une région répétée de 39 nucléotides a démontré que les races affichaient la même fréquence chez les isolats collectés de 1991 à 1999, tandis que 95% appartenaient à la race Ct0 en 2010, probablement parce que les variétés de lentilles sont réceptives à la race Ct0, mais qu’environ un tiers des variétés étaient résistantes à Ct1.

Keywords:

• anthracnose
• Colletotrichum lentis
• Colletotrichum truncatum
• epidemiology
• fungicide decision support system
• intergenic spacer (IGS)
• lentil
• molecular markers
• pathogen races
• resistance

Mots clés:

• anthracnose
• Colletotrichum truncatum
• Colletotrichum lentis
• épidémiologie
• espaceur intergénique (IGS)
• lentille
• marquers moléculaires
• races d’agents pathogènes
• résistance
• système d’aide à la décision relatif à l’application des fongicides

Introduction

Currently, between 1 and 1.5 million hectares are seeded to lentil (Lens culinaris subsp. culinaris Medic) in western Canada. The majority of the harvested seed are destined for human consumption in India, Turkey and countries in Europe and South America. In 1987, a fungal pathogen causing typical anthracnose symptoms and serious yield losses in lentil was identified in the province of Manitoba (Morrall, 1988). The pathogen was initially named Colletotrichum truncatum (Schwein.) Andrus & W.D., but was renamed C. lentis (Damm) in 2014 (Damm et al., 2014). Over the next few years, the disease also was found in the provinces of Saskatchewan and Alberta (Morrall, 1997), and later in neighbouring North Dakota, USA (Venette et al., 1994). Lentil anthracnose has occasionally been reported in other countries, including Bulgaria (Kaiser et al., 1998), Bangladesh, Ethiopia and Syria (Morrall, 1997). It continues to be the most serious disease of Canadian-grown lentil. Annual disease surveys in Saskatchewan between 2012 and 2015 showed that anthracnose was present in 60–83% of lentil fields (Dokken-Bouchard et al., 2016). The majority of published research on this disease has been generated by scientists in western Canada. The present review summarizes studies on epidemiology, fungicide control, C. lentis races, identification of resistant lentil lines, inheritance of resistance genes, molecular markers linked to resistance, and the recent discovery that polymorphisms in ribosomal DNA differentiate pathogen races.

Longevity of C. lentis

Initial research into lentil anthracnose was conducted at the University of Manitoba where both the disease cycle and epidemiology of C. lentis were elucidated (Buchwaldt et al., 1996). It was demonstrated that the pathogen forms microsclerotia on infected stems and leaflets during the growing season (Buchwaldt, 2011). Microsclerotia consist of cells with high melamine content which reduce the physical effects of repeated wetting and drying, fluctuating temperatures and degradation by microorganisms in the soil. The microsclerotia enable the pathogen to survive on lentil debris after harvest and serve as a primary source of inoculum in subsequent lentil crops. A study of longevity showed that C. lentis remained viable for 3 years when infected lentil debris was buried in the soil, but caused less infection after 1 year when left on the soil surface exposed to weather extremes (Buchwaldt et al., 1996). Since minimal tillage is common in western Canada, this practise inadvertently helps reduce the primary source of inoculum level in fields. This study led to conclusions that lentil anthracnose can be partially managed by reduced tillage and a rotation of 3–4 years between lentil crops.

Long distance dispersal of C. lentis

Initial research established the importance of windborne inoculum for spread of C. lentis between fields. It was shown that microsclerotia on lentil debris, dust and soil particles are dispersed to neighbouring fields by wind, particularly during combining of infected crops (Buchwaldt et al., 1996). When these materials were overwintered outdoors, they caused infection when inoculated onto lentil plants the following year. Consequently, new lentil crops are at risk of infection from previous anthracnose-infected crops in nearby fields. In contrast, spread of C. lentis by seedborne infection is less important, supported by the fact that several studies have not been able to demonstrate transmission of the pathogen from infected seeds to germinating seedlings under field conditions (Gibson, 1993; Morrall, 1997). Taken together, it appears that wind dispersed inoculum contribute to spread of anthracnose in lentil producing areas of North America, while seedborne infection is relatively unimportant.

Disease cycle of C. lentis

Anthracnose symptoms typically develop on lentil plants at the 10–12 node stage or at early flowering and can be seen as tan coloured necrotic lesions on lower leaflets and stems which are closest to the soilborne inoculum. Infected leaflets drop to the soil surface. This premature leaf drop indicates that fungicide treatment is needed to protect the remainder of the crop, as described in more detail below. The pathogen is polycyclic and new generations of conidia continuously form in lesions on leaves and stems. Secondary spread occurs when conidia are splashed by rain to the upper canopy, to neighbouring plants or even further by blowing rain (Buchwaldt, 2011). Under optimal climatic conditions, the pathogen can spread about 30 metres from a single infection site in the field in a matter of a few weeks (L. Buchwaldt, unpublished).

Optimal time of fungicide application

Ascochyta blight caused by Ascochyta lentis (Vassiljevsky) also is an important foliar disease of lentil in western Canada. Inoculum of both anthracnose and ascochyta blight builds up in short lentil rotations particularly in wet growing seasons. In order to reduce yield losses caused by the two diseases, fungicides were tested to obtain efficacy data needed for their registration. The active ingredients included chlorothalonil and azoxystrobin. Field trials demonstrated that the optimal time of application is the 10–12 node stage to early flowering, with a second application at mid flowering which is 10–14 days later (Buchwaldt et al., 1999; Chongo et al., 1999). Yield increases ranged from 300 to 800 kg per hectare which were well above the economic threshold. The second application was economically beneficial under high disease pressure. The optimal time for application of fungicides containing chlorothalonil and azoxystrobin continue to be implemented in the annual ‘Guide to Crop Protection’ produced by the Government of Saskatchewan (2017a).

Fungicide decision support system

Yield losses caused by anthracnose and ascochyta blight and high cost of fungicides created a need for a fungicide decision support system (FDSS) to be used by lentil growers and staff in public and private extension services. A FDSS was designed around four risk factors: (A) plant density, (B) number of days with rain in the last 14 days, (C) rain in the 5-day weather forecast and (D) early symptoms of anthracnose and ascochyta blight (Table 1). Plant density is positively correlated with yield but negatively correlated with disease severity; consequently, fungicide cost is more likely to be recovered in crops with high plant density. The number of rain events in the weeks leading up to the fungicide decision, as well as the likelihood of rain in the weather forecast, is important for initial infection by both anthracnose and ascochyta. Identification of early disease symptoms, particularly the premature leaf-drop caused by anthracnose, indicates that primary inoculum is present in the field (Fig. 1a). Importantly, fungicide application is effective at this point in time as it controls the secondary spread of conidia, thereby reducing the rate of disease development in the field. Between 0 and 30 points were assigned to each risk factor, resulting in a range of total risk points between 0 and 85. To determine the threshold value for fungicide application, the FDSS was evaluated in 20 field tests and five lentil varieties over 3 years (Supplementary Table S1, field test 1–20). Fungicides with two different active ingredients (a.i.) were tested: chlorothalonil at 1 kg a.i. ha⁻¹ and azoxystrobin at 125 g a.i. ha⁻¹. The fungicides were applied at early and mid-flower, at which time the total risk points were calculated in each field test. The final disease severity was rated before harvest and yield data were collected. Data analysis showed that eight field tests with 50–65 total risk points corresponded to a significant yield increase from fungicide application (Supplementary Table S1, field test 1–8). Concurrently, 12 field tests with 10–40 total risk points corresponded to an absence of yield increase in nine field tests (Supplementary Table S1, field test 9–17); but a yield increase was obtained in three field tests (Supplementary Table S1, field test 18–20). Taken together, when 50 risk points was used as the threshold value for fungicide treatment, the FDSS resulted in 85% correct assessments (17 of 20 field tests). The fungicide decision support system was part of a website ‘Pulse Crop Diseases’ available from 2000 to 2005, that also provided growers and extension staff in the public and private sectors with knowledge about diagnosis of early disease symptoms, epidemiology and control options for the major diseases of pulse crops in western Canada. Several other communication tools were used to disseminate this information including newsletters, articles in grower magazines, field days and grower meetings. The FDSS and related information were incorporated into web pages of the Saskatchewan Ministry of Agriculture and Saskatchewan Pulse Growers and in their ‘Pulse Production Manual’ (2000) (see http://proof.saskpulse.com/files/general/150625_Fungicide_Decision_Support_Checklist_for_control_of_Ascochyta_Blight_and_Anthracnose_in_Lentil3.pdf.)

Table 1. Fungicide decision support system for control of anthracnose (C. lentis) and ascochyta blight (A. lentis) of lentil in western Canada. The threshold value for fungicide application is 50 total risk points.

	Risk points
A. Plant density
Thin (high weed pressure, low yield expectation)	0
Moderate (some weeds, possibly low yield)	5
Normal (about 12 plants per square foot)	10
Dense (more plants than normal, lush growth)	15
B. Number of days with rain in the last 14 days
0 days	0
1–2 days	5
3–4 days	10
5–6 days	15
7 days or more	20
C. Rain in the 5-day weather forecast
Dry	0
Unpredictable	10
Light showers	15
Rain	20
D. Symptoms of anthracnose and ascochyta blight
No visible symptoms	0
Few lesions on lower ½ of the foliage (up to 10% of the leaflets infected)	5
Lesions on lower ½ of foliage (up to 25% of the leaflets infected)	15
Lesions on lower as well as upper foliage	25
Lesions on lower foliage and premature leaf drop characteristic of anthracnose	25
Flowers and/or peduncles infected, characteristic of ascochyta blight	25
Lesions on the stem base	30
Total risk points	From 0 to 85

Fig. 1 (Colour online) a, Premature leaf drop in lentil indicating the presence of initial inoculum of Colletotrichum lentis in the field. b, Lentil plant with lesions on stems and leaflets. c, Close-up of a leaf lesion with microsclerotia, setae and acervuli of C. lentis.

Host range of C. lentis

Colletotrichum lentis infects both Lens and Vicia (vetch) species and causes typical anthracnose symptoms on all above-ground plant parts whether in the field or under controlled conditions in a growth cabinet (Gossen et al., 2009). The necrotic lesions are sunken and surrounded by a dark margin, and readily support formation of acervuli containing secondary conidia and setae (Fig. 1b). In contrast, Pisum sativum L. (pea) and Cicer arietinum L. (chickpea) develop few superficial lesions that do not sporulate under field conditions, while more distantly related legume species such as Lupinus albus L. (lupine), Medicago sativa L. (alfalfa) and Glycine max (L.) Merr. (soybean) do not develop symptoms under any environmental conditions (Gossen et al., 2009).

In Manitoba, where C. lentis was first discovered, lentil was initially produced under contract by a limited number of growers, many of which planted the crop in short rotation due to high commodity prices. As a result, lentils were grown intensively in a relatively small area. However, it is unlikely that C. lentis was introduced with the planted seed from outside the province, since the disease is almost absent in other countries, and the majority of seed lots for planting were produced locally. Thus, C. lentis most likely arose as a host shift from another Colletotrichum species, possibly on local wild vetches such as Vicia americana Muhl. ex Willd. or cultivated species such as Vicia fabae L. (faba bean) grown on a small scale in Manitoba. Such host shifts might occur in plant pathogens that can adapt to crop species introduced into new geographical areas as described in a review by Silva et al. (2012).

Identification of C. lentis

Fungal plant pathogen species are defined by their host range, disease symptoms, morphology of conidia and other fungal structures, along with DNA polymorphisms in coding as well as non-coding genomic regions. The pathogen that causes lentil anthracnose forms single-celled slightly falcate conidia (3–6 × 18–30 µm) with one truncated and one tapered end (Buchwaldt, 2010). The conidia are formed in acervuli interspersed with black setae (3.5–8.0 × 60–120 µm). Microsclerotia, measuring less than 1 mm, consist of several hundred melanized cells that ensure the pathogen’s survival outside the host plant (Buchwaldt et al., 1996). The anamorph stage of the lentil pathogen in western Canada was originally identified as C. truncatum (Schwein.) Andrus & W.D. Moore (Morrall, 1988). In 2014, it was renamed C. lentis Damm based on its host specialization on Lens and Vicia species (Gossen et al., 2009), morphology of conidia, and sequence of ITS (internal transcribed spacer) region of the ribosomal DNA (Damm et al., 2014). A teleomorph stage consisting of perithecia and single-celled ascospores has been induced in the laboratory and was tentatively named Glomerella truncata sp. nov. (Armstrong-Cho & Banniza, 2006); however, it has not been found under natural conditions.

Anthracnose resistance screening method

A method has been developed for screening of lentil germplasm accessions for resistance to anthracnose (Buchwaldt et al., 2004). Single spore isolates of C. lentis were obtained from infected lentil plants or seed samples from commercial fields in Manitoba and Saskatchewan. Cultures were maintained on half strength oatmeal agar (Difco, USA) in 9 cm Petri dishes incubated at 23°C in a 16 h photoperiod. Long-term storage at −80°C was achieved by placing 7 mm plugs from actively growing cultures in cryo-freezer solution (10% skim milk, 40% glycerol). Inoculum needed for inoculation was produced by transferring plugs from −80°C to half strength oatmeal agar in Petri plates which were incubated for 7–10 days at 23°C in a 16 h photoperiod. Conidia were obtained by flooding the cultures with sterile water followed by filtering through Mira cloth (Calbiochem, USA) and adjusting to 100 000 conidia per mL water. Lentil plants at the early flowering stage were spray-inoculated with the conidial suspension, incubated at 100% relative humidity for 24 h, and subsequently transferred to a greenhouse. Disease symptoms were rated 10–14 days after inoculation as follows: The number of lesions on stems was counted in increments of five, and their appearance noted as either superficial, deeply penetrating, or a mixture of both. The degree of plant wilt was rated as none, shoot die-back (one side branch wilted), half, or the entire plant wilted. It was not possible to rate leaf infection because of variable degrees of leaf drop due to competition for light, and the physical effect of watering and moving of plants. Plants with only superficial stem lesions up to 30 were considered resistant, and plants with a mixture of 0–10 superficial and deep stem lesions were moderately resistant; in both cases there were no signs of shoot die-back or plant wilt. For a detailed description of the anthracnose screening method see Buchwaldt et al. (2004) and Shaikh et al. (2013).

Resistance in Lens culinaris to races of C. lentis

A search was initiated for anthracnose resistance in cultivated lentil (L. culinaris) by screening of accessions obtained worldwide. Seed samples were acquired through several gene banks, including the Vavilov Institute of Plant Industry in St Petersburg, Russia (prefix VIR), the US Department of Agriculture-Agriculture Research Services (USDA) in Pullman, WA, USA (prefix PI), Institute for Plant Genetics and Plant Research in Gartersleben, Germany (prefix LENS), and ICARDA (International Center for Agricultural Research in the Dry Areas) in Aleppo, Syria (prefix ILL). More than 2300 L. culinaris accessions obtained from 50 countries were screened using the method described above. In 2000, a subset of 357 lentil accessions was deposited in the Canadian gene bank and was given the prefix CN (Plant Gene Resources of Canada; Buchwaldt & Richards, 2004). Four years later, results from anthracnose screening of 1772 lentil accessions were included in the USDA’s Germplasm Resources Information Network GRIN (U.S. National Plant Germplasm System). Resistant accessions such as PI299331 (Chile), PI320937 (Germany) and PI345629 (former Soviet Union) became a resource for studies of the infection process, fungicide efficacy, genetics of inheritance, and identification of molecular markers linked to resistance described in this review. Importantly, resistance in ‘Indianhead’, PI320937 and PI345629 were the same whether screened in the field or under controlled conditions in a growth chamber (Chongo & Bernier, 1999a). However, many gene bank accessions segregated for anthracnose severity either due to heterozygosity at resistance loci, or because the original accession was a mixture of slightly different genotypes. This necessitated several cycles of inoculation followed by selfing of single resistant plants and re-testing of progenies, after which homozygous resistant lines were generated from the original accessions (Buchwaldt et al., 2004; Shaikh et al., 2013). Development of resistant lentil lines congruently allowed identification of races of the C. lentis pathogen. So far, two races, Ct0 and Ct1, have been identified in western Canada based on differential disease reaction of lentil lines (Buchwaldt et al., 2004; Shaikh et al., 2013). Three L. culinaris lines – VIR2633 (Georgia), VIR2058 and VIR2076 (Czech Republic) – have resistance to both race Ct0 and Ct1, while six lines have resistance to race Ct0 (Fig. 2). In addition, 49 lines have resistance to race Ct1.

Fig. 2 (Colour online) Cultivated lentil lines (Lens culinaris) in different seed market classes selected for resistance to Colletotrichum lentis race Ct0 and Ct1, the cause of anthracnose in western Canada.

Infection process of C. lentis

Microscopic examination of the infection process revealed that C. lentis is a hemi-biotrophic pathogen on lentil. Conidia germinate 3–6 h post inoculation (hpi), appressoria form 6–12 hpi, and infection pegs penetrate the epidermal cells directly (Chongo et al., 2002). Hyphae grow both inter- and intra-cellular during a symptomless phase lasting 72 h, followed by a necrotic and cell-destroying phase beginning 3–6 days after inoculation. The rate of disease development is affected by temperature. The optimum conditions for infection are 20 and 24°C with 24 h leaf wetness (Chongo & Bernier, 1999b). Under these conditions, new lesions form in 7 days (incubation period) and conidia are produced after 13 days (latent period). In contrast, temperatures under 15°C greatly reduce the rate of disease development. In resistant lines, such as PI320937, phenolic compounds accumulate at penetration sites, resulting in slower hyphal growth, and fewer and smaller lesions compared with a susceptible line (Chongo et al., 2002).

Anthracnose resistance in Canadian lentil varieties

A few years after discovery of lentil anthracnose in western Canada, variety ‘Indianhead’, also known as plant introduction PI320952, was identified as being resistant to this disease (Bernier et al., 1992; Gibson, 1993). However, its black seed coat colour makes it unsuitable for human consumption. Consequently, a crossing programme to transfer anthracnose resistance into lentil with appropriate seed characteristics was initiated in collaboration with the Crop Development Centre (CDC) at the University of Saskatchewan, the only lentil breeding programme in Canada (Buchwaldt et al., 1995). The first anthracnose resistant variety was ‘CDC Robin’ (Vandenberg et al., 2002) followed by ‘CDC Redberry’ (Vandenberg et al., 2006) and several others. New lentil varieties in western Canada are routinely screened for resistance to race Ct1, and about one-third of lentil varieties have some resistance to this race (Government of Saskatchewan, 2017b).

Genetics of anthracnose resistance

A genetic study of inheritance showed that resistance to race Ct1 in L. culinaris ‘Indianhead’, PI320937 and PI345629 is conferred by a combination of recessive and dominant genes. So far, two recessive genes, ctr1 and ctr2, and three dominant genes, CtR3, CtR4 and CtR5, have been identified (Buchwaldt et al., 2013). Progenies in F₁, F₂, BC₁ and F₃ derived from crosses between each of the resistant lines and the susceptible cultivar, ‘Eston’, demonstrate that ‘Indianhead’ has one recessive and one dominant gene (ctr1, CtR3), PI345629 also has one recessive and one dominant gene (ctr2, CtR5) that are different from those in ‘Indianhead’, while PI320937 has a single dominant gene (CtR4) that is different from the others (Buchwaldt et al., 2013). Segregation ratios in F₂ from intercrossing the three resistant lines showed that the resistance genes are closely linked and likely different alleles at a single locus. The genetics of resistance to race Ct0 in L. culinaris has not yet been examined.

Anthracnose resistance in wild lentil species

Resistance to race Ct1 and Ct0 was also sought in six wild lentil species, Lens ervoides (Brign) Grande, L. lamottei Czefr, L. nigricans (Bieb) Godron, L. odemensis L., L. orientalis Boiss and L. tomentosus L. Accessions with resistance to both races were identified in Lens ervoides (Tullu et al., 2006a). Since wild lentil species have late flowering, low yield and unsuitable seed morphology, it was necessary to transfer resistance to cultivated lentil. A single resistant L. ervoides accession, PI72847, was therefore crossed with the susceptible L. culinaris variety ‘Eston’. Rescue of ovules from F₁ pods was required because of crossing barriers between wild and cultivated species (Fiala et al., 2009). A single F₁ hybrid plant was obtained which was selfed to produce a population of 85 F₈ lines. Disease screening showed 25% of these lines were resistant to race Ct0 and 22% to race Ct1, while none were resistant to both races. The authors concluded that two recessive genes likely conferred resistance, but warned the results could be skewed due to high progeny mortality during population development. However, it is also worth noting that inoculation of hybrid progenies was carried out with only 3000 and 10 000 conidia per mL water of race Ct0 and Ct1, respectively, not 100 000 conidia per mL used for screening of L. culinaris described above. In a similar study, another resistant L. ervoides line, IG72815, was crossed with ‘Eston’ followed by embryo rescue of F₁ hybrid progenies. In the resulting F₈ population, 29% of lines were resistant to race Ct0, again indicating that resistance might be conferred by two recessive genes (Tullu et al., 2013). However, in this study, the conidial concentration used for inoculation of hybrid progenies was not stated. The authors describe that in addition to the crossing barrier between L. culinaris and L. ervoides, there were problems in subsequent generations with sterility, inability of seed to germinate and poor plant growth. The F₈ population consisted of lines with extremely small seed size, reduced plant height, late flowering and indeterminate vegetative growth.

Molecular markers linked to C. lentis and A. lentis resistance

A number of studies have focused on identification of molecular markers in L. culinaris linked to anthracnose and ascochyta blight resistance loci and their effectiveness in marker-assisted selection (MAS) of breeding lines. Initially, Tullu et al. (2003) identified two RAPD markers, OPE06₁₂₅₀ in repulsion and UBC-704₇₀₀ in coupling, which flanked the resistance locus in PI320937 using a bi-parental F₆ population derived from a cross with ‘Eston’. These markers also were linked to a resistance locus in variety ‘Indianhead’. The resistance loci are most likely CtR4 in PI320937 and CtR3 in ‘Indianhead’ described in the genetic study above (Buchwaldt et al., 2013). Simultaneously, Tar’an et al. (2003) demonstrated the usefulness of marker-assisted selection using a bi-parental F₇ population from a cross between varieties ‘CDC Robin’ (derived from ‘Indianhead’) with anthracnose resistance and breeding line 946a-46 (derived from accession ILL5588) with ascochyta resistance (Ford et al., 1999). Three RAPD markers were used to genotype the F₇ progenies, OP06₁₂₅₀ linked in repulsion to a locus for anthracnose resistance, and UBC227₁₂₉₀ and RB18₆₈₀ linked in coupling to two different ascochyta resistance loci (Ford et al., 1999). A total of 85% of anthracnose resistant progenies were correctly identified based on the absence of OP06₁₂₅₀. Similarly, 74% and 80% of ascochyta resistant progenies were correctly selected based on the presence of either UBC2271₂₉₀ or RB18₆₈₀ and when inoculated with two different A. lentis isolates. These results demonstrated the usefulness of MAS for pyramiding ascochyta and anthracnose resistance that could be used in a breeding programme.

Accession PI320937 was initially known for anthracnose resistance, and was later shown to also have ascochyta blight resistance. Subsequently, a bi-parental F₈ population derived from a cross between PI320937 and ‘Eston’ was phenotyped for ascochyta blight reaction and genotyped with RAPD and AFLP markers. Analysis of quantitative trait loci (QTL) identified a locus on linkage group six explaining 33% (LOD 7.8) of ascochyta blight resistance (Tullu et al., 2006b). Interestingly, the ascochyta resistance QTL contained RAPD marker OPE06₁₂₅₀, which is linked to anthracnose resistance described above (Tar’an et al., 2003; Tullu et al., 2003), demonstrating a hot spot of genes on linkage group six conferring resistance to both diseases in L. culinaris.

IGS polymorphisms in C. lentis related to race structure

It is well known that Colletotrichum and many other fungal species can be differentiated at the genomic level by polymorphisms in certain genes as well as the intergenic spacer (IGS) and internal transcribed spacer (ITS) regions of ribosomal DNA (Cannon et al., 2012). We identified both length and single-nucleotide polymorphisms in two repeat-rich regions of the IGS consisting of 23 and 39 nucleotides (nt) that almost perfectly distinguish isolates of the two C. lentis races (Durkin et al., 2015). Both length and DNA sequence variations within the 23 nt repeat separate Ct1 isolates having 17 repeats of identical sequence from Ct0 isolates having either 14 or 19 repeats of different sequence variations (Durkin et al., 2015). A length polymorphism in the 39 nt repeat separated Ct1 isolates having 7 or 9 repeats from Ct0 isolates having only two or four repeats. This clear length polymorphism was used to survey C. lentis isolates collected from 1991 to 1999 and again in 2010 in the provinces of Saskatchewan and Manitoba (Durkin et al., 2015). The number of 39 nt repeats among isolates showed that the two races were present at equal frequency in the first period, while race Ct0 comprised 95% of the pathogen population decades later. Seeding of Ct1 resistant lentil varieties, which began around 2001, likely contributed to lowering the frequency of race Ct1 in the pathogen population, while at the same time, the frequency of Ct0 increased because all lentil varieties are susceptible to this race.

Discussion

Colletotrichum lentis is a serious disease problem of lentil in western Canada and parts of Northern USA, but is only sporadically reported in Europe and South America. Consequently, most research on this disease was undertaken by scientists in Canada. Epidemiological studies showed that microsclerotia form on infected lentil plants and survive up to 3 years in the soil, but for less time when left on the soil surface. The microsclerotia are the primary source of inoculum, while secondary spread within the field occurs by rain splash of conidia formed in acervuli on infected plants. The pathogen is spread between fields as windborne microsclerotia on infected plant debris and soil, while seedborne infection is less important. Taken together, lentil growers are recommended to follow a 3- or 4-year crop rotation, practice reduced tilling, and seed new lentil fields at a distance from previously anthracnose-infected crops. In addition, growers can use a fungicide decision support system to assess whether fungicide application at early to mid-flower is likely to result in a yield increase. The FDSS is based on four risk factors, including rainfall and early disease symptoms (Table 1), especially the premature leaf drop caused by C. lentis (Fig. 1B).

Screening of a world collection of L. culinaris germplasm resulted in identification of 58 (2.5%) accessions with anthracnose resistance (Buchwaldt et al., 2004; Shaikh et al., 2013). Simultaneously, two C. lentis races, Ct0 and Ct1, were identified based on their differential disease reaction on a subset of accessions (Fig. 2). Ct1 resistance in variety ‘Indianhead’ was successfully transferred to Canadian lentil varieties by traditional crossing at the Crop Development Centre, University of Saskatchewan, and about one-third of current varieties have resistance derived from this source (Government of Saskatchewan). Furthermore, it was demonstrated that marker-assisted selection was useful for combining resistance to anthracnose caused by C. lentis race Ct1 and ascochyta blight caused by A. lentis (Tar’an et al., 2003; Tullu et al., 2003).

The two C. lentis races can be differentiated based on DNA polymorphisms in the intergenic spacer region of ribosomal DNA (Durkin et al., 2015). Length polymorphisms of a 39 nt repeat in the IGS region was utilized in a survey of isolates which showed that race Ct0 comprise 95% of the pathogen population in Saskatchewan in 2010. This helps explain why most Canadian lentil varieties were highly susceptible to anthracnose in the 2011 and 2012 growing seasons, where most of the tested varieties reached 95–100% disease severity, except for ‘CDC Imigreen’ and ‘CDC Viceroy’ which reached 41% and 53% severity, respectively (Wunsch & Schatz, 2011; Wunsch et al., 2012). It can be concluded that although Canadian varieties are routinely screened for Ct1 resistance, their field performance is quite different due to the dominance of race Ct0. Fortunately, resistance to race Ct0 exists in both L. culinaris (Buchwaldt et al., 2004; Shaikh et al., 2013) and L. ervoides (Tullu et al., 2006a). Not surprisingly, transfer of resistance from L. ervoides to L. culinaris resulted in low recovery of hybrids with acceptable agronomic traits, since crossing outside the cultivated gene pool creates a linkage drag of undesirable genes, making it difficult to restore yield, agronomic traits and seed in acceptable market classes. In addition, it is worth noting that resistance to Ct0 in L. ervoides only was detectable at 3% of the spore concentration used to screen L. culinaris (Fiala et al., 2009). Conversely, an opportunity exists to develop new varieties with resistance to Ct0 by utilizing one or more of the nine L. culinaris lines that have seed sizes and seed coat colours resembling accepted market classes (Fig. 2). Yellow seeded lentil with green seed coat colour is consumed directly, while red lentil with tan seed coat first undergoes dehulling and splitting. Transfer of resistance by traditional crossing within L. culinaris lines is relatively easy, and has already been achieved for Ct1 resistance. Furthermore, the recently published genome sequence of L. culinaris makes identification of single nucleotide polymorphic markers linked to Ct0 resistance feasible (Sharpe et al., 2013). Although PGRC maintain L. culinaris accessions from around the world (Buchwaldt & Richards, 2004), seed of the anthracnose resistant lines (Fig. 2) that were generated by selection over multiple cycles of inoculation and selfing of single resistant plants showing resistance to one or both C. lentis races are available from the corresponding author.

Acknowledgements

We are grateful for support from technicians in the Department of Plant Science, University of Manitoba, at Agriculture and Agri-Food Canada in Saskatoon, Scott, Swift Current, Indian Head, Canora, Redvers and Outlook. We also value the cooperation of many lentil producers in both Manitoba and Saskatchewan. The photos in Fig. 2 were produced by Patricia Breakey and Lori Jones-Flory.

Supplemental material

Supplemental data for this article can be accessed online here: https://doi.org/10.1080/07060661.2018.1441185.

Additional information

Funding

Research grants to the Department of Plant Science, University of Manitoba came from the Manitoba Pulse Growers Association and the Natural Sciences and Engineering Research Council of Canada (NSERC). Research grants to Agriculture and Agri-Food Canada, Saskatoon Research and Development Centre came from the Saskatchewan Pulse Growers and Saskatchewan Agri-Food Innovation Fund (AFIF).