Association mapping reveals a reciprocal virulence/avirulence locus within diverse US Pyrenophora teres f. maculata isolates.

BACKGROUND: Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an economically important disease of barley that also infects wheat. Using genetic analysis to characterize loci in Ptm genomes associated with virulence or avirulence is an important step to identify pathogen effectors that determine compatible (virulent) or incompatible (avirulent) interactions with cereal hosts. Association mapping (AM) is a powerful tool for detecting virulence loci utilizing phenotyping and genotyping data generated for natural populations of plant pathogenic fungi. RESULTS: Restriction-site associated DNA genotyping-by-sequencing (RAD-GBS) was used to generate 4,836 single nucleotide polymorphism (SNP) markers for a natural population of 103 Ptm isolates collected from Idaho, Montana and North Dakota. Association mapping analyses were performed utilizing the genotyping and infection type data generated for each isolate when challenged on barley seedlings of thirty SFNB differential barley lines. A total of 39 marker trait associations (MTAs) were detected across the 20 barley lines corresponding to 30 quantitative trait loci (QTL); 26 novel QTL and four that were previously mapped in Ptm biparental populations. These results using diverse US isolates and barley lines showed numerous barley-Ptm genetic interactions with seven of the 30 Ptm virulence/avirulence loci falling on chromosome 3, suggesting that it is a reservoir of diverse virulence effectors. One of the loci exhibited reciprocal virulence/avirulence with one haplotype predominantly present in isolates collected from Idaho increasing virulence on barley line MXB468 and the alternative haplotype predominantly present in isolates collected from North Dakota and Montana increasing virulence on barley line CI9819. CONCLUSIONS: Association mapping provided novel insight into the host pathogen genetic interactions occurring in the barley-Ptm pathosystem. The analysis suggests that chromosome 3 of Ptm serves as an effector reservoir in concordance with previous reports for Pyrenophora teres f. teres, the causal agent of the closely related disease net form net blotch. Additionally, these analyses identified the first reported case of a reciprocal pathogen virulence locus. However, further investigation of the pathosystem is required to determine if multiple genes or alleles of the same gene are responsible for this genetic phenomenon.

First report of rubbery rot of potato caused by Geotrichum candidum in the United States.

Rubbery rot of potato caused by Geotrichum candidum Link is characterized by symptoms of damp, flaccid tubers that feel rubbery when squeezed (Humpreys-Jones 1969), similar in consistency to potato diseases such as pink rot (caused by Phytophthora erythroseptica) and Pythium leak (caused by species of Pythium). In November 2019, several symptomatic tubers of potato variety 'Ciklamen' that had been held in storage since harvest and originated from an over-head irrigated, sandy-loam production field in Bingham county, Idaho were submitted to the University of Idaho for diagnosis. Shipping-point inspection records indicated 4-9% of tubers were affected. External symptoms included irregularly shaped, randomly located sunken black-colored lesions on more severely affected rubbery-textured tubers. When cut, internal affected tissue developed a greyish appearance after several minutes. Lens-shaped cavities were apparent in two of the tubers, indicating an advanced infection. A sour-milk smell accompanied the sample. To isolate the pathogen, pieces of tuber tissue approximately 5 mm in diameter were collected from the margins of symptomatic areas and surface-sanitized in sodium hypochlorite (2%) for two minutes, rinsed twice in sterile water and plated onto tap water agar amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After three days at 21°C, colonies having distinct creamy white mycelia, a sweet, juniper-like odor, and hyaline hyphae were consistently associated with diseased tissue. Cylindrical to oval-shaped arthroconidia ranged in size from 6.6-11.0 × 3.2-5.9 µm (mean = 8.4 × 4.7, n=21), within dimensions as reported by Carmichael (1957). No other pathogens including species of Pythium and Phytophthora were recovered from the sample. Pure cultures were obtained by transferring hyphal tips to potato dextrose agar plates. Species identity was confirmed via rDNA ITS sequencing using primers ITS5/4 (White et al., 1990). DNA extraction and PCR conditions were as previously described (Woodhall et al., 2013). Resulting sequences (NCBI accession numbers MT893312 and MT893315) shared 99.4% identity with G. candidum Accession KY103453.1 on GenBank. To confirm pathogenicity, ten tubers (cv. Ciklamen) were inoculated by placing a 10mm2 plug of fully colonized PDA of G. candidum on the tuber surface, and ten tubers were mock inoculated with sterile PDA plugs. After 27 days at 21C in a dew chamber, tubers were examined for symptoms. Eight of the 10 inoculated tubers exhibited a rubbery texture and fluid leaking from tubers when cut, with two tubers also exhibiting a grey internal discoloration and the distinctive smell. Control tubers did not exhibit any symptoms. Isolations were attempted from four symptomatic tubers and G. candidum was successfully recovered from three tubers. The disease has been reported sporadically in the United Kingdom (Humphreys-Jones 1969) and Korea (Kim et al., 2011) and the pathogen occurs worldwide (Carmichael 1957). Though the fungus causes a tomato rot in the United States (US) (Pritchard and Porte, 1923; Bourret et al., 2013), and potatoes with rubbery rot originating from Australia were intercepted at a US port (Farr et al., 2020), the disease has not to our knowledge been documented on potato grown in the US. Because symptoms may be confused with pink rot and Pythium leak, it is critical for producers to obtain a correct diagnosis to facilitate appropriate management strategies.