Field Pea (Pisum sativum) Germplasm Screening for Seedling Ascochyta Blight Resistance and Genome-Wide Association Studies Reveal Loci Associated with Resistance to Peyronellaea pinodes and Ascochyta koolunga.

Ascochyta blight is a damaging disease that affects the stems, leaves, and pods of field pea (Pisum sativum) and impacts yield and grain quality. In Australia, field pea Ascochyta blight is primarily caused by the necrotrophic fungal species Peyronellaea pinodes and Ascochyta koolunga. In this study, we screened 1,276 Pisum spp. germplasm accessions in seedling disease assays with a mix of three isolates of P. pinodes and 641 accessions with three mixed isolates of A. koolunga (513 accessions were screened with both species). A selection of three P. sativum accessions with low disease scores for either pathogen, or in some cases both, were crossed with Australian field pea varieties PBA Gunyah and PBA Oura, and recombinant inbred line populations were made. Populations at the F3:4 and F4:5 generation were phenotyped for their disease response to P. pinodes and A. koolunga, and genotypes were determined using the diversity arrays technology genotyping method. Marker-trait associations were identified using a genome-wide association study approach. Trait-associated loci were mapped to the published P. sativum genome assembly, and candidate resistance gene analogues were identified in the corresponding genomic regions. One locus on chromosome 2 (LG1) was associated with resistance to P. pinodes, and the 8 Mb genomic region contains 156 genes, two of which are serine/threonine protein kinases, putatively contributing to the resistance trait. A second locus on chromosome 5 (LG3) was associated with resistance to A. koolunga, and the 35 Mb region contains 488 genes, of which five are potential candidate resistance genes, including protein kinases, a mitogen-activated protein kinase, and an ethylene-responsive protein kinase homolog.

The novel avirulence effector AlAvr1 from Ascochyta lentis mediates host cultivar specificity of ascochyta blight in lentil.

Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little is known about the molecular mechanism of disease or host specificity. We employed a map-based cloning approach using a biparental A. lentis population to clone the gene AlAvr1-1 that encodes avirulence towards the lentil cultivar PBA Hurricane XT. The mapping population was produced by mating A. lentis isolate P94-24, which is pathogenic on the cultivar Nipper and avirulent towards Hurricane, and the isolate AlKewell, which is pathogenic towards Hurricane but not Nipper. Using agroinfiltration, we found that AlAvr1-1 from the isolate P94-24 causes necrosis in Hurricane but not in Nipper. The homologous corresponding gene in AlKewell, AlAvr1-2, encodes a protein with amino acid variation at 23 sites and four of these sites have been positively selected in the P94-24 branch of the phylogeny. Loss of AlAvr1-1 in a gene knockout experiment produced a P94-24 mutant strain that is virulent on Hurricane. Deletion of AlAvr1-2 in AlKewell led to reduced pathogenicity on Hurricane, suggesting that the gene may contribute to disease in Hurricane. Deletion of AlAvr1-2 did not affect virulence for Nipper and AlAvr1-2 is therefore not an avirulence gene for Nipper. We conclude that the hemibiotrophic pathogen A. lentis has an avirulence effector, AlAvr1-1, that triggers a hypersensitive resistance response in Hurricane. This is the first avirulence gene to be characterized in a legume pathogen from the Pleosporales and may help progress research on other damaging Ascochyta pathogens.

Agrobacterium tumefaciens-mediated transformation and expression of GFP in Ascochyta lentis to characterize ascochyta blight disease progression in lentil.

The plant immune system is made up of a complex response network that involves several lines of defense to fight invading pathogens. Fungal plant pathogens on the other hand, have evolved a range of ways to infect their host. The interaction between Ascochyta lentis and two lentil genotypes was explored to investigate the progression of ascochyta blight (AB) in lentils. In this study, we developed an Agrobacterium tumefaciens-mediated transformation system for A. lentis by constructing a new binary vector, pATMT-GpdGFP, for the constitutive expression of green fluorescent protein (EGFP). Green fluorescence was used as a highly efficient vital marker to study the developmental changes in A. lentis during AB disease progression on the susceptible and resistant lentil accessions, ILL6002 and ILL7537, respectively. The initial infection stages were similar in both the resistant and susceptible accessions where A. lentis uses infection structures such as germ tubes and appressoria to gain entry into the host while the host uses defense mechanisms to prevent pathogen entry. Penetration was observed at the junctions between neighbouring epidermal cells and occasionally, through the stomata. The pathogen attempted to penetrate and colonize ILL7537, but further fungal advancement appeared to be halted, and A. lentis did not enter the mesophyll. Successful entry and colonization of ILL6002 coincided with structural changes in A. lentis and the onset of necrotic lesions 5-7 days post inoculation. Once inside the leaf, A. lentis continued to grow, colonizing all parts of the leaf followed by plant cell collapse. Pycnidia-bearing spores appeared 14 days post inoculation, which marks the completion of the infection cycle. The use of fluorescent proteins in plant pathogenic fungi together with confocal laser scanning microscopy, provide a valuable tool to study the intracellular dynamics, colonization strategy and infection mechanisms during plant-pathogen interaction.