Identification and characterisation of potential sources of resistance to Ascochyta blight within the exotic germplasm of lentil

Abstract

Lentil (Lens culinaris Medikus. ssp. culinaris) is high value cool season legume staple food and cash crop, cultivated globally including in Australia. However, the crop is often challenged by biotic and abiotic stresses that reduce the full yield potential. Among these, Ascochyta blight (AB) caused by Ascochyta lentis affects gross profits and yield stability in Australia. Resistant cultivars are the most viable, long-term sustainable option among the recommended disease management strategies. However, the susceptibility of previously released resistant cultivars (Northfield and Nipper) and a future uncertainty over the resistance status of the few remaining available resistance sources has necessitated an immediate influx of novel and diverse resistance sources into the Australian lentil breeding program. To aid in this, the potential of exotic germplasm of lentil including landraces and wild species to provide such new resistance to AB has been evaluated.

To the time, space and financial commitment, a focused identification of germplasm strategy (FIGS) was applied to aid in the identification of novel AB resistance sources from the lentil landrace collection of the International Center for Agricultural Research in Dry Areas (ICARDA), Morocco. This entailed a systemic filtering of germplasm collection sites against environmental variables favouring AB progression to predict regions harbouring AB resistant genotypes. Accordingly, a subset (87 accessions) with the highest probabilities for harbouring AB resistance was selected from a collection of 4576 accessions. The subset was tested against a highly aggressive Australian isolate of A. lentis (FT13037) in a completely randomised and replicated bioassay, which resulted in identification of a highly resistant accession (IG 207) and twelve (IG 96, IG 712, IG 914, IG 1687, IG 1735, IG 5911, IG 7104, IG 7593, IG 7731, IG 8218, IG 8360 and IG 8550) moderately resistant accessions. Additionally, screening of 30 accessions from five-wild species against aggressive isolates (FT13037 and FT13038) revealed two highly resistant Lens orientalis accessions (ILWL 180 and ILWL 7) and a ten (L. nigricans (6), L. odomensis (1), L. ervoides (1), L. lamottei (1), and L. orientalis (1)) moderately resistant accessions. Subsequent screening of the resistant accessions IG 207 (L. culinaris) and ILWL 180 (L. orientalis) against a group of geographically diverse isolates with variable level of aggressiveness revealed superior resistance of both accessions over current resistance sources such as ILL 7537 and Indianhead.

Replicated histopathological studies of the lentil/A. lentis pathosystem validated the results of the bioassays and the AB resistance of IG 207 and ILWL 180. These are proposed to be, more effective than existing sources through strong and early activation of defence mechanisms. Further evidence of higher levels of reactive oxygen species (ROS; hydrogen peroxide and superoxide) and phenolic compounds from ILWL 180 in response to AB infection added to this conclusion.

Introgression of novel and diverse resistance genes into the cultivated genetic background will likely improve lentil resistance to AB and minimise yield losses. Therefore, an interspecific cross between ILWL 180 and ILL 6002 was attempted. Despite, being a wild relative, ILWL 180 was crossed with the cultivar ILL 6002 and produced healthy F1 seeds. The segregation of the subsequent F2 population indicated two recessive genes confer resistance to AB. Additionally, the genetic analysis of other desirable traits within the F2 population revealed beneficial variants for traits such as days to first flower, plant height at flowering, number of nodes below first flower node, seed diameter, 100-seed weight and seed yield that may be helpful in widening the genetic base of cultivated lentil.

To select the physical location of the gene effects and possible candidate genes on the genome, a linkage map was constructed using 815 high quality single nucleotide polymorphism (SNP) markers generated from a transcriptome sequencing of the F5 recombinant inbred line (RIL) population (N = 140). The map stretched 488.02 centiMorgan (cM) along eight linkage groups (LGs) with an 0.66 cM average marker-marker distance. Genetic dissection of the RIL population detected a quantitative trait loci (QTL) on LG5 which harbored nine putative candidate genes linked to AB resistance. Of these, five candidate genes were directly related to plant defence responses. Moreover, two of the three nonsynonymous mutations within the coding sequences of the putative candidate genes (Uroporphyrinogen decarboxylase (UROD) and Glutathione - S – transferase, DHAR3, chloroplastic (GST-DHAR3)) were predicted to modify the 3D structure of the corresponding proteins and ultimately the phenotype of the cultivar ILL 6002. Overall, the disease symptomatology, physiological and biochemical responses, and genetic evidence of resistance against AB infection support the conclusion that a stable and novel AB resistance was identified and characterised from ILWL 180 that offers significant potential to improve AB resistance of the existing cultivars within the Australian lentil breeding program.