Detection of Podosphaera macularis in Air Samples by Quantitative PCR.

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay uses primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit of the nuclear ribosomal DNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

Identification of quantitative trait loci associated with R1-mediated resistance to powdery mildew and sex determination in hop (Humulus lupulus L.).

KEY MESSAGE: Two QTL were identified using linkage mapping approaches, one on hop linkage group 3 (qHl_Chr3.PMR1) associated with powdery mildew resistance and a second on linkage group 10 (cqHl_ChrX.SDR1) associated with sex determination. Hop (Humulus lupulus L.) is a dioecious species cultivated for use in beer. Hop powdery mildew, caused by Podosphaera macularis, is a constraint in many growing regions. Thus, identifying markers associated with powdery mildew resistance and sex provides the opportunity to pyramid R-genes and select female plants as seedlings, respectively. Our objectives were to characterize the genetic basis of R1-mediated resistance in the cultivar Zenith which provides resistance to pathogen races in the US, identify quantitative trait loci (QTL) associated with R1 and sex, and develop markers for molecular breeding-based approaches. Phenotypic evaluation of the population indicated that R1-based resistance and sex are inherited monogenically. We constructed a genetic map using 1339 single nucleotide polymorphisms (SNPs) based upon genotype-by-sequencing of 128 F1 progeny derived from a Zenith × USDA 21058M biparental population. SNPs were assigned to 10 linkage groups comprising a map length of 1204.97 cM with an average density of 0.94 cM/marker. Quantitative trait locus mapping identified qHl_Chr3.PMR1, associated with R1 on linkage group 3 (LOD = 23.57, R2 = 57.2%), and cqHl_ChrX.SDR1, associated with sex on linkage group 10 (LOD = 5.42, R2 = 25.0%). Kompetitive allele-specific PCR (KASP) assays were developed for both QTL and assessed against diverse germplasm. Our results indicate that KASP markers associated with R1 may be limited to materials that are pedigree-related to Zenith, whereas markers associated with sex may be transferable across populations. The high-density map, QTL, and associated KASP markers will enable selecting for sex and R1-mediated resistance in hop.

Impact of Infection Timing and Autumnal Fungicide Applications on Perennation of Pseudoperonospora humuli and Severity of Hop Downy Mildew.

Pseudoperonospora humuli, causal agent of hop downy mildew, is known to survive winter as systemic mycelium in the crown and developing buds of hop (Humulus lupulus). Field studies were conducted over three growing seasons to quantify the association of infection timing to overwintering of P. humuli and development of downy mildew. Cohorts of potted plants were inoculated sequentially from early summer to autumn, overwintered, and then evaluated for symptoms of systemic downy mildew in emerging shoots. Shoots with systemic P. humuli developed after inoculation at any time in the previous year, with the most severe disease typically resulting from inoculation in August. Independent of the timing of inoculation, diseased shoots emerged coincident with the emergence of healthy shoots, beginning as early as late February and continuing through late May to early June. Surface crown buds on inoculated plants exhibited internal necrosis associated with P. humuli at rates ranging from 0.3 to 1.2%, whereas P. humuli was detected by PCR on 7.8 to 17.0% of asymptomatic buds depending on the timing of inoculation and year. Four experiments were conducted to quantify the impact of foliar fungicides applied in autumn on downy mildew the following spring. There was a small reduction of disease in only one study. Together, these studies indicate that infection by P. humuli that leads to overwintering can occur over a broad period of time, but delaying infection until autumn tends to reduce disease levels in the following year. However, in established plantings, postharvest application of foliar fungicides appeared to have little impact on severity of downy mildew in the ensuring year.