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 utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. 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.

Temperature Influences on Powdery Mildew Susceptibility and Development in the Hop Cultivar Cascade.

The hop cultivar 'Cascade' possesses partial resistance to powdery mildew (Podosphaera macularis) that can be overcome by recently emerged, virulent isolates of the fungus. Given that hop is a long-lived perennial and that brewers still demand Cascade, there is a need to better understand factors that influence the development of powdery mildew on this cultivar. Growth chamber experiments were conducted to quantify the effect of constant, transient, and fluctuating temperature on Cascade before, concurrent to, and after inoculation as contrasted with another powdery mildew-susceptible cultivar, 'Symphony'. Exposure of plants to supraoptimal temperature (26 and 32°C) before inoculation led to more rapid onset of ontogenic resistance in intermediately aged leaves in Cascade as compared with Symphony. Cascade was overall less susceptible to powdery mildew when exposed to constant temperature ranging from 18 to 32°C directly after inoculation. However, cultivar also interacted with temperature such that proportionately fewer and smaller colonies developed on Cascade than Symphony at supraoptimal yet permissive temperatures for disease. When plants were inoculated and then exposed to high temperature, colonies became progressively more tolerant to temperatures of 26 to 30°C with increasing time from inoculation to exposure, as moderated by cultivar, the specific temperature, and their interaction. Subjecting plants to simulated diurnal temperature regimes at the time of inoculation or 24 h later indicated Cascade and Symphony responded proportionately similarly on days predicted to be marginally unfavorable or marginally favorable for powdery mildew, although Cascade was quantitatively less susceptible than Symphony. In sum, this research indicates that Cascade is overall less susceptible to powdery mildew than Symphony, and supraoptimal temperature before, concurrent to, or after infection may interact differentially to moderate disease risk in Cascade. Therefore, cultivar-specific risk assessments for powdery mildew appear warranted.

First report of Powdery Mildew Caused by Golovinomyces ambrosiae on Cannabis sativa in Oregon.

Oregon is the second largest producer of hemp in the United States with 25,900 ha of hemp licensed to growers in 2019, a nearly six-fold increase over the previous year (Perkowski 2019, Capital Press). Industrial hemp has a wide range of uses including textiles to nutritional supplements; in Oregon, hemp has become one of the most economically promising crops and is mainly cultivated for cannabidiol (CBD) production. Between 2018 and 2019, multiple independent greenhouse growers in western Oregon reported powdery mildew-like signs and symptoms on leaves and buds of several Cannabis sativa cultivars, including 'Cherry Wine'. Signs of the disease started as small, white, powdery patches, typically on the adaxial sides of leaves, and progressed to coalescent colonies on leaves, stems, and buds. Fungi present on diseased tissues had unbranched hyaline conidiophores that measured 140 to 250 µm and grew erect from caulicolous and amphigenous mycelium (n = 15). Foot cells were cylindrical, often tapered at one or both ends, and measured 80 to 117 × 9.5 to 11.9 µm (n = 15). Conidia were catenescent, hyaline, ellipsoidal to barrel-shaped, lacked fibrosin bodies, and measured 24 to 34 × 12 to 18 µm (n = 50). No chasmothecia were observed. Morphological observations overlapped with several Golovinomyces spp. Including G. ambrosiae, G. cichoracearum, and G. spadiceus (Braun and Cook 2012). Identification was confirmed by bidirectional sequencing and phylogenetic analysis of 1,457 nucleotides from the concatenated internal transcribed spacer (ITS), 28S large ribosomal subunit, and beta-tubulin (TUB2) regions of two isolates using primer pairs ITS1/ITS4 and NL1/LR5, and TubF1/TubR1 respectively (Mori et al. 2000, Qiu et al. 2020, Vilgalys and Hester 1990, White et al. 1990; GenBank Acc. No.: MW248121 to MW248124, MW265971 to MW265972). The Oregon hemp isolates grouped (bootstrap value = 100) in a monophyletic clade with G. ambrosiae accessions from Qiu et al. (2020). Pathogenicity was confirmed by transferring conidia by leaf rub inoculation onto 2-to 4-week-old 'Cherry Wine' potted plants and incubated outdoors at 12 to 22°C. Control plants were mock-inoculated using healthy leaves. Powdery mildew symptoms developed on inoculated plants approximately 14 to 21 days later; control plants were asymptomatic. Identification was confirmed by morphological characterization and sequencing using the aforementioned primers. The hemp isolates were also able to infect detached leaves of Humulus lupulus 'Symphony' via similar inoculations; however, colony development on 'Symphony' was slow and sporulation sparse as was reported by Weldon et al. (2020). Golovinomyces spp. have also been reported on hemp in Kentucky (Szarka et al. 2019), Ohio (Farinas and Peduto Hand 2020), and New York (Weldon et al. 2020). Although reported as G. spadiceus, these reports are also likely G. ambrosiae according to new taxonomic revision of the genus (Qiu et al. 2020). This is the first known report of Golovinomyces ambrosiae causing powdery mildew on hemp in Oregon (OSC 171893). While powdery mildew on hemp currently appears most severe in protected cultivation, rapid expansion of hemp cultivation and introduction of new CBD varieties throughout Oregon could lead to increased powdery mildew risk in outdoor cultivation.

Development of a Diagnostic Assay for Race Differentiation of Podosphaera macularis.

Hop powdery mildew (caused by Podosphaera macularis) was confirmed in the Pacific Northwest in 1996. Before 2012, the most common race of P. macularis was able to infect plants that possessed powdery mildew resistance based on the R-genes Rb, R3, and R5. After 2012, two additional races of P. macularis were discovered that can overcome the resistance gene R6 and the partial resistance found in the cultivar Cascade. These three races now occur throughout the region, which can complicate management and research efforts because of uncertainty on which race(s) may be present in the region and able to infect susceptible hop genotypes. Current methods for determining the races of P. macularis are labor intensive, costly, and typically require more than 14 days to obtain results. We sought to develop a molecular assay to differentiate races of the fungus possessing virulence on plants with R6, referred to as V6-virulent, from other races. The transcriptomes of 46 isolates of P. macularis were sequenced to identify loci and variants unique to V6 isolates. Fourteen primer pairs were designed for 10 candidate loci that contained single nucleotide polymorphisms (SNP) and short insertion-deletion polymorphisms. Two differentially labeled locked nucleic acid probes were designed for a contig that contained a conserved SNP associated with V6-virulence. The resulting duplexed real-time PCR assay was validated against 46 V6 and 54 non-V6 P. macularis isolates collected from the United States and Europe. The assay had perfect discrimination of V6-virulence among isolates of P. macularis originating from the western U.S. but failed to predict V6-virulence in three isolates collected from Europe. The specificity of the assay was tested with different species of powdery mildew fungi and other microorganisms associated with hop. Weak nonspecific amplification occurred with powdery mildew fungi collected from Vitis vinifera, Fragaria sp., and Zinnia sp.; however, nonspecification amplification is not a concern when differentiating pathogen race from colonies on hop. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.

Population Diversity and Structure of Podosphaera macularis in the Pacific Northwestern United States and Other Populations.

Powdery mildew, caused by Podosphaera macularis, is one of the most important diseases of hop. The disease was first reported in the Pacific Northwestern United States, the primary hop-growing region in this country, in the mid-1990s. More recently, the disease has reemerged in newly planted hopyards of the eastern United States, as hop production has expanded to meet demands of local craft brewers. The spread of strains virulent on previously resistant cultivars, the paucity of available fungicides, and the potential introduction of the MAT1-2 mating type to the western United States, all threaten sustainability of hop production. We sequenced the transcriptome of 104 isolates of P. macularis collected throughout the western United States, eastern United States, and Europe to quantify genetic diversity of pathogen populations and elucidate the possible origins of pathogen populations in the western United States. Discriminant analysis of principal components grouped isolates within three to five geographic populations, dependent on stringency of grouping criteria. Isolates from the western United States were phenotyped and categorized into one of three pathogenic races based on disease symptoms generated on differential cultivars. Western U.S. populations were clonal, irrespective of pathogenic race, and grouped with isolates originating from Europe. Isolates originating from wild hop plants in the eastern United States were genetically differentiated from all other populations, whereas isolates from cultivated hop plants in the eastern United States mostly grouped with isolates originating from the west, consistent with origins from nursery sources. Mating types of isolates originating from cultivated western and eastern U.S. hop plants were entirely MAT1-1. In contrast, a 1:1 ratio of MAT1-1 and MAT1-2 was observed with isolates sampled from wild plants or Europe. Within the western United States a set of highly differentiated loci were identified in P. macularis isolates associated with virulence to the powdery mildew R-gene R6. The weight of genetic and phenotypic evidence suggests a European origin of the P. macularis populations in the western United States, followed by spread of the pathogen from the western United States to re-emergent production regions in the eastern United States. Furthermore, R6 compatibility appears to have been selected from an extant isolate within the western United States. Greater emphasis on sanitation measures during propagation and quarantine policies should be considered to limit further spread of novel genotypes of the pathogen, both between and within production areas.

Evolution of the U.S. Biological Select Agent Rathayibacter toxicus.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Evolutionary transitions between beneficial and phytopathogenic Rhodococcus challenge disease management.

Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.