QTL dissection and mining of candidate genes for Ascochyta fabae and Orobanche crenata resistance in faba bean (Vicia faba L.)

Background Ascochyta blight caused by Ascochyta fabae Speg. and broomrape (Orobanche crenata) are among the economically most significant pathogens of faba bean. Several QTLs conferring resistance against the two pathogens have been identified and validated in different genetic backgrounds. The aim of this study was to saturate the most stable QTLs for ascochyta and broomrape resistance in two Recombinant Inbred Line (RIL) populations, 29H x Vf136 and Vf6 x Vf136, to identify candidate genes conferring resistance against these two pathogens. Results We exploited the synteny between faba bean and the model species Medicago truncatula by selecting a set of 219 genes encoding putative WRKY transcription factors and defense related proteins falling within the target QTL intervals, for genotyping and marker saturation in the two RIL populations. Seventy and 50 of the candidate genes could be mapped in 29H x Vf136 and Vf6 x Vf136, respectively. Besides the strong reduction of the QTL intervals, the mapping process allowed replacing previous dominant and pedigree-specific RAPD flanking markers with robust and transferrable SNP markers, revealing promising candidates for resistance against the two pathogens. Conclusions Although further efforts in association mapping and expression studies will be required to corroborate the candidate genes for resistance, the fine-mapping approach proposed here increases the genetic resolution of relevant QTL regions and paves the way for an efficient deployment of useful alleles for faba bean ascochyta and broomrape resistance through marker-assisted breeding.

Didymella fabae (leaf and pod spot).

Ascochyta blight is the most severe disease of cool-season pulses (Davidson and Kimber, 2007). D. fabae (anamorph: Ascochyta fabae) attacks Vicia faba and can survive and reproduce in and spread from crop debris or be transported in infected seed. Introduction on infected seed occurred in Australia and Canada in the 1970s, and was probably the means for the pathogen's original spread to countries outside southwestern Asia. Ascospores are disseminated by wind from the debris as primary inoculum and secondary cycles are initiated by conidia spread by rain splash from plant lesions. The fungus is host-specific in causing disease, but may be able to survive in non-host plants and reproduce on their debris. It is not treated as a phytosanitary risk or listed as an invasive pathogen by major organizations. Seed certification is the primary means of preventing its spread to new areas and the importation of new genotypes of the fungus to areas already infested.

Identification of a Polyketide Synthase Gene Responsible for Ascochitine Biosynthesis in Ascochyta fabae and Its Abrogation in Sister Taxa

ABSTRACT The polyketide-derived secondary metabolite ascochitine is produced by species in the Didymellaceae family, including but not restricted to Ascochyta species pathogens of cool-season food legumes. Ascochitine is structurally similar to the well-known mycotoxin citrinin and exhibits broad-spectrum phytotoxicity and antimicrobial activities. Here, we identified a polyketide synthase (PKS) gene (denoted pksAC) responsible for ascochitine production in the filamentous fungus Ascochyta fabae. Deletion of the pksAC prevented production of ascochitine and its derivative ascochital in A. fabae. The putative ascochitine biosynthesis gene cluster comprises 11 genes that have undergone rearrangement and gain-and-loss events relative to the citrinin biosynthesis gene cluster in Monascus ruber. Interestingly, we also identified pksAC homologs in two recently diverged species, A. lentis and A. lentis var. lathyri, that are sister taxa closely related to ascochitine producers such as A. fabae and A. viciae-villosae. However, nonsense mutations have been independently introduced in coding sequences of the pksAC homologs of A. lentis and A. lentis var. lathyri that resulted in loss of ascochitine production. Despite its reported phytotoxicity, ascochitine was not a pathogenicity factor in A. fabae infection and colonization of faba bean (Vicia faba L.). Ascochitine was mainly produced from mature hyphae at the site of pycnidial formation, suggesting a possible protective role of the compound against other microbial competitors in nature. This report highlights the evolution of gene clusters harnessing the structural diversity of polyketides and a mechanism with the potential to alter secondary metabolite profiles via single nucleotide polymorphisms in closely related fungal species. IMPORTANCE Fungi produce a diverse array of secondary metabolites, many of which are of pharmacological importance whereas many others are noted for mycotoxins, such as aflatoxin and citrinin, that can threaten human and animal health. The polyketide-derived compound ascochitine, which is structurally similar to citrinin mycotoxin, has been considered to be important for pathogenicity of legume-associated Ascochyta species. Here, we identified the ascochitine polyketide synthase (PKS) gene in Ascochyta fabae and its neighboring genes that may be involved in ascochitine biosynthesis. Interestingly, the ascochitine PKS genes in other legume-associated Ascochyta species have been mutated, encoding truncated PKSs. This indicated that point mutations may have contributed to genetic diversity for secondary metabolite production in these fungi. We also demonstrated that ascochitine is not a pathogenicity factor in A. fabae. The antifungal activities and production of ascochitine during sporulation suggested that it may play a role in competition with other saprobic fungi in nature.

QTLs for ascochyta blight resistance in faba bean (Vicia faba L.): validation in field and controlled conditions

Ascochyta blight is an important disease of faba bean (Vicia faba L.). Yield losses can be as high as 90% and losses of 35–40% are common. The line 29H is one of the most resistant accessions to the pathogen (Ascochyta fabae Speg.) ever described. In this work, we aimed to validate across generations the main quantitative trait loci (QTLs) for ascochyta blight resistance identified in the cross 29H × Vf136 and to test their stability under field conditions. QTLs located on chromosomes II and III have been consistently identified in the recombinant inbred line (RIL) population of this cross, in both controlled (growth chamber) and field conditions and, thus they are good targets for breeding. In addition, a new QTL for disease severity on pods has been located on chromosome VI, but in this case, further validation is still required. A synteny-based approach was used to compare our results with previous QTL works dealing with this pathogen. Our results suggest that the QTL located on chromosome II, named Af2, is the same one reported by other researchers, although it is likely that the donors of resistance differ in the allele conferring the resistance. By contrast, the location of Af3 on chromosome III does not overlap with the position of Af1 reported by other authors, suggesting that Af3 may be an additional source of resistance to ascochyta blight.