Cynthia M. Ocamb

Benzimidazole resistance offusarium species recovered from potatoes with dry rot from storages located in the Columbia basin of oregon and Washington

Fusarium species cause dry rot or seedpiece decay of potato tubers in storage and in the field. When potato storages were sampled in the Columbia Basin of Oregon and Washington,F. sambucinum, F. oxysporum, andF. solani were the predominantFusarium species recovered from potato tubers with dry rot. OtherFusarium species recovered includedF. avenaceum, F. culmorum, F. equiseti, F. proliferatum, andF. sporotrichioides. The majority ofF. sambucinum isolates tested were resistant to thiabendazole at 5, 10, and 25 mg liter-1 in amended potato dextrose agar (PDA), whereas resistance was less frequent inF. oxysporum andF. solani isolates. When isolates ofF. sambucinum, F. solani, andF. oxysporum were grown on PDA with no thiabendazole, the average colony diameter of isolates with resistance to thiabendazole was greater than the average colony diameter of isolates sensitive to thiabendazole for each respective species. When isolates ofF. sambucinum were placed on tuber slices, lesions were apparent by four days after inoculation, and lesion diameters were generally greater than those associated withF. solani orF. oxysporum. At eight days after inoculation onto tuber slices, the average lesion diameter ofF. sambucinum, F. solani, orF. oxysporum isolates with resistance to thiabendazole was greater than the average lesion diameter of isolates sensitive to thiabendazole for each respective species. Thiabendazole-resistance was also detected inF. avenaceum, F. culmorum, F. equiseti, andF. sporotrichioides.ResumenLas especies deFusarium causan pudrición seca del tubérculo de papa o pudriciones en el almacén y el campo. Cuando se hizo un muestreo en la Cuenca Columbia de Oregon y Washington,F. sambucinum, F. oxysporum yF. solani fueron las especies predominates extraídas de tubérculos de papa con pudrición seca. Otras especies extraídas incluyeronF. avenaceum, F. culmorum, F. equiseti, F. proliferatum yF. sporotrichioides. La mayoría de aislamientos probados deF. sambucinum fueron resistentes al thiabendazole en a 5, 10 y 25mg/L-1 en papa-dextrosa-agar (PDA) modificado, mientras que la resistencia fue menos frecuente en aislamientos deF. oxysporum yF. solani. Cuando se sembraron aislamientos deF. sambucinum, F. solani yF. oxysporum en PDA sin thiabendazole, el diámetro promedio de la colonia de los aislamientos con resistencia a thiabendazole fue mayor que el diámetro promedio de los aislamientos sensibles al thiabendazole para la especie respectiva. Cuando los aislamientos deF. sambucinum fueron colocados en rodajas de tubérculo, las lesiones fueron visibles cuatro días después de la inoculación y el diámetro de las lesiones fue mayor que aquel asociado conF. solani o conF. oxysporum. A los ocho de la inoculación en rodajas, el diámetro promedio de la lesión de los aislamientos deF. sambucinum, F. solani oF. oxysporum con resistencia a thiabendazole fue mayor que el diámetro promedio de los aislamientos sensibles a este producto para cada especie. También se detectó resistencia al thiabendazole enF. avenaceum, F. culmorum, F. equiseti yF. sporotrichioides.

Genetic and Pathogenic Relatedness of Pseudoperonospora cubensis and P. humuli

The most economically important plant pathogens in the genus Pseudoperonospora (family Peronosporaceae) are Pseudoperonospora cubensis and P. humuli, causal agents of downy mildew on cucurbits and hop, respectively. Recently, P. humuli was reduced to a taxonomic synonym of P. cubensis based on internal transcribed spacer (ITS) sequence data and morphological characteristics. Nomenclature has many practical implications for pathogen identification and regulatory considerations; therefore, further clarification of the genetic and pathogenic relatedness of these organisms is needed. Phylogenetic analyses were conducted considering two nuclear and three mitochondrial loci for 21 isolates of P. cubensis and 14 isolates of P. humuli, and all published ITS sequences of the pathogens in GenBank. There was a consistent separation of the majority of the P. humuli isolates and the P. cubensis isolates in nuclear, mitochondrial, and ITS phylogenetic analyses, with the exception of isolates of P. humuli from Humulus japonicus from Korea. The P. cubensis isolates appeared to contain the P. humuli cluster, which may indicate that P. humuli descended from P. cubensis. Host-specificity experiments were conducted with two reportedly universally susceptible hosts of P. cubensis and two hop cultivars highly susceptible to P. humuli. P. cubensis consistently infected the hop cultivars at very low rates, and sporangiophores invariably emerged from necrotic or chlorotic hypersensitive-like lesions. Only a single sporangiophore of P. humuli was observed on a cucurbit plant during the course of the studies. Together, molecular data and host specificity indicate that there are biologically relevant characteristics that differentiate P. cubensis and P. humuli that may be obfuscated if P. humuli were reduced to a taxonomic synonym of P. cubensis. Thus, we recommend retaining the two species names P. cubensis and P. humuli until the species boundaries can be resolved unambiguously.

First Report of Hop Powdery Mildew in the Pacific Northwest

Hop powdery mildew (HPM) was first observed in commercial hop (Humulus lupulus L.) fields in Washington State on 10 June 1997 near Toppenish in the Yakima Valley. The disease appeared throughout the valley in 1997; by mid-July, scattered fields throughout the Yakima growing area reported HPM. Approximately 2,000 of 30,000 acres in production were not harvested in 1997 due to HPM. The pathogen apparently perennated in buds, and flagshoots originating from infected buds were observed during March and April 1998 at various locations throughout the Yakima Valley. During the 1999 growing season, the majority of hop acreage in Washington State was affected, and most fields planted to susceptible cultivars contained at least one infected plant. HPM was initially discovered in southern Idaho during early July 1998, in two adjacent fields of hops in Canyon County. HPM was found ≈644 km (400 miles) north in another hop-growing region of Idaho, Boundary County, during mid-July 1998. HPM eventually was observed in more than 20 Idaho hop fields. The initial discovery of HPM in Oregons Willamette Valley was made during late-July 1998, in two neighboring hop fields. By the end of the growing season, HPM was observed in nine commercial fields representing 3.7% of the hop production acreage in Oregon. Affected cultivars include Brewers Gold, Chinook, Cluster, Columbus/Tomahawk, Eroica, Fuggle, Galena, Golding, Liberty, Olympic, Perle, Symphony, Tettnanger, Willamette, and Zeus. Infected basal leaves of bines had small whitish circular spots on adaxial surfaces. In some cases, blisters preceded direct observation of the fungus. Cones also were infected, appearing stunted and malformed. The pathogen usually was visible on infected cones but sometimes was found only under overlapping bracts. Cleistothecia have not been observed in the field to date. Conidia were transferred to leaf disks (12 mm diameter) excised from greenhouse-grown cv. Galena hop plants. Inoculated leaf disks were incubated on moistened filter paper in glass petri dishes at 20°C with illumination provided for a 12 h day by two cool-white fluorescent bulbs. HPM lesions with chains of unicellular, barrel-shaped conidia (30 to 36 × 15 to 18 μm) were visible within 7 days. The causal agent was identified as Sphaerotheca macularis (Wallr.:Fr.) Lind (synonym S. humuli (DC.) Burrill) on the basis of conidial shape and size as well as host range (1). Reference: (1) D. J. Royle. 1978. Powdery mildew of the hop. Pages 381-409 in: The Powdery Mildews. D. M. Spencer, ed. Academic Press, New York.

Predicting Infection Risk of Hop by Pseudoperonspora humuli

ABSTRACT Downy mildew, caused by Pseudoperonospora humuli, is one of the most destructive diseases of hop. Weather factors associated with infection risk by P. humuli in the maritime region of western Oregon were examined for 24- and 48-h periods and quadratic discriminant function models were developed to classify periods as favorable for disease development on leaves. For the 24-h data sets, the model with superior predictive ability included variables for hours of relative humidity>80%, degree-hours of wetness, and mean night temperature. The same variables were selected for the 48-h data sets, with the addition of a product variable for mean night temperature and hours of relative humidity>80%. Cut-points (pT) on receiver operating characteristic curves that minimized the overall error rate were identified by selecting the cut-point with the highest value of Youdens index. For the 24- and 48-h models these were pT=0.49 and 0.39, respectively. With these thresholds, the sensitivity and specificity of the models in cross validation by jackknife exclusion were 83.3 and 88.8% for the 24-h model and 87.5 and 84.4% for the 48-h model, respectively. Cut-points that minimized the average costs associated with disease control and crop loss due to classification errors were determined using estimates of economic damage during vegetative development and on cones near harvest. Use of the 24- and 48-h models was estimated to reduce average management costs during vegetative development when disease prevalence was <0.31 and 0.16, respectively. Using economic assumptions near harvest, management decisions informed by the models reduced average costs when disease prevalence was <0.21 and 0.1 for the 24- and 48-h models, respectively. The value of the models in management decisions was greatest when disease prevalence was relatively low during vegetative development, which generally corresponds to the normally drier period from late spring to midsummer in the Pacific Northwest of the United States.

Forecasting and Management of Hop Downy Mildew

Downy mildew of hop (Humulus lupulus), caused by Pseudoperonospora humuli, is managed in the Pacific Northwestern United States by regular application of fungicides. A degree-day model that forecasts the first emergence of shoots systemically infected with P. humuli (termed basal spikes) and a risk index for secondary spread of the disease were evaluated over four seasons in western Oregon. In surveys conducted in 34 hop yards, the predicted first spike emergence occurred on average 11.6 days (median 12 days) after spike emergence using a simple average degree-day model (base temperature 6.5°C) developed for Washington State. Predictions based on a single sine model (base temperature 6°C) provided on average 4.9 days (median -0.5 days) of advanced warning before the first spike emerged. Downy mildew severity in a previous season was negatively correlated with the degree-day emergence date of spikes the following year (r = -0.39). In experimental plots, disease severity was significantly greater where fungicide applications were timed using a risk index compared to routine fungicide applications in 2005 and 2007, but statistically similar between these treatments in 2006 and 2008. However, in 2006, 2007, and 2008, treatments initiated using a degree-day threshold resulted in an area under the disease progress curve similar to or smaller than in treatments with routine fungicide applications. Model-aided treatments required four fewer fungicide applications compared to routine fungicide applications. These studies indicate that downy mildew can be managed effectively with fewer fungicide applications than currently made by hop growers in this region if fungicide applications are timed to coincide with the predicted emergence of basal spikes and subsequent disease risk forecasts.

Powdery Mildew Outbreaks caused by Podosphaera macularis on Hop Cultivars Possessing the Resistance Gene R6 in the Pacific Northwestern United States

Resistant cultivars of hop (Humulus lupulus) have been grown, with the aim of helping to manage powdery mildew in the Pacific Northwest since the first report of the disease in the field in 1997 (4). A major objective of many breeding programs is development of resistance to powdery mildew, and this has generally been achieved by single resistance genes (qualitative resistance). One such gene, R6 (3), has been utilized extensively in new cultivars and has prevented epidemics of the disease in those cultivars across the Pacific Northwestern United States for approximately 15 years. In 2011, a grower in Washington State reported outbreaks of powdery mildew on cv. Apollo, which is thought to possess powdery mildew resistance derived from R6. Fungicides and cultural control measures were applied, and the grower reported no substantial crop damage from the disease. During the winter of 2012, the same grower planted rhizomes of cv. Apollo in a greenhouse in the Yakima Valley of Washington State and later found the plants to be affected by powdery mildew. Affected leaves from plants of cvs. Apollo, Newport, and Nugget (all reported [3] or assumed to possess R6 based on pedigree) grown in the same greenhouse were later provided to the authors. Conidia obtained from each affected plants were transferred to plants of the highly susceptible cv. Symphony, which is not known to contain any resistance genes. After 10 to 14 days of incubation, resultant conidia from each cultivar above (total of three isolates) were transferred to greenhouse grown plants of cvs. Nugget and Symphony and incubated at 18°C. Within 7 days, all three isolates produced powdery mildew colonies characteristic of P. macularis (2) on both cultivars. Cleistothecia did not develop in any colonies. In addition, Nugget and Symphony plants were inoculated with a field population of P. macularis originating from cultivars lacking R6 in Oregon. These inoculations on Nugget did not develop powdery mildew whereas Symphony plants did. Non-inoculated controls remained free of powdery mildew. Results were identical in two additional experiments. The sequence of the mating type idiomorph, MAT1-1, was obtained to confirm identity of the pathogen as P. macularis as described previously (1). The sequences were identical among the three isolates obtained from the greenhouse in Washington and isolates of P. macularis obtained previously from Oregon and Washington. MAT1-2 idiomorph was not detected in the isolates collected. While R6-virulent strains have been detected previously in race characterization experiments, these strains have not caused widespread epidemics of powdery mildew. The increasing prevalence of virulent strains of P. macularis and outbreaks of powdery mildew on formerly resistant cultivars necessitates changes in breeding strategies and disease management efforts to minimize damage resulting from the disease. The distribution of virulent strains of the pathogen and susceptibility of formerly resistance cultivars to powdery mildew are currently under investigation. References: (1) B. Asalfet et al. Phytopathology 103:717, 2013. (2) R. Bélanger et al. The Powdery Mildews: a Comprehensive Treatise. APS Press, St. Paul, MN, 2002. (3) P. Darby. Brew Hist. 121:94, 2005. (4) C. Ocamb et al. Plant Dis. 83:1072, 1999.

Quantitative assessment of Anguina sp. and Rathayibacter rathayi in Dactylis glomerata seed production fields in oregon and estimates of yield loss

Anguina sp. is a nematode that infests the inflorescence of orchardgrass and forms galls that replace the seed. Anguina sp. is also a vector of the bacterial pathogen Rathayibacter rathayi, which causes galls or gummosis in orchardgrass (Dactylis glomerata) panicles. The percentage of orchardgrass panicles infected or percentage of seed loss from Anguina sp. or R. rathayi in five commercial orchardgrass seed-production fields in Oregon during 2003 and 2004 was determined. The percentage of panicles with Anguina sp. ranged from 9 to 24%, although the percentage of seed replaced by Anguina sp. was less than 0.2%. The number of galls per panicle ranged from 1 to 29. However, more than 50% of Anguina-infested panicles contained only a single Anguina gall and few panicles had eight or more galls. The percentage of panicles with R. rathayi ranged from 3 to 27%. Percentage of seed loss from R. rathayi ranged from 0.1 to 7.3%. Seed loss in orchardgrass seed-production fields assessed for both Anguina sp. and R. rathayi was found to be as great as 8%. The number of Anguinagalls remaining in fields following harvest ranged from 0 to 40 per square meter.

Distribution and Characterization of Podosphaera macularis Virulent on Hop Cultivars Possessing R6-Based Resistance to Powdery Mildew

Host resistance, both quantitative and qualitative, is the preferred long-term approach for disease management in many pathosystems, including powdery mildew of hop (Podosphaera macularis). In 2012, an epidemic of powdery mildew occurred in Washington and Idaho on previously resistant cultivars whose resistance was putatively based on the gene designated R6. In 2013, isolates capable of causing severe disease on cultivars with R6-based resistance were confirmed in Oregon and became widespread during 2014. Surveys of commercial hop yards during 2012 to 2014 documented that powdery mildew is now widespread on cultivars possessing R6 resistance in Washington and Oregon, and the incidence of disease is progressively increasing. Pathogenic fitness, race, and mating type of R6-virulent isolates were compared with isolates of P. macularis lacking R6 virulence. All isolates were positive for the mating type idiomorph MAT1-1 and were able to overcome resistance genes Rb, R3, and R5 but not R1 or R2. In addition, R6-virulent isolates were shown to infect differential cultivars reported to possess the R6 gene and also the R4 gene, although R4 has not yet been broadly deployed in the United States. R6-virulent isolates were not detected from the eastern United States during 2012 to 2015. In growth chamber studies, R6-virulent isolates of P. macularis had a significantly longer latent period and produced fewer lesions on plants with R6 as compared with plants lacking R6, indicating a fitness cost to the fungus. R6-virulent isolates also produced fewer conidia when compared with isolates lacking R6 virulence, independent of whether the isolates were grown on a plant with or without R6. Thus, it is possible that the fitness cost of R6 virulence occurs regardless of host genotype. In field studies, powdery mildew was suppressed by at least 50% on plants possessing R6 as compared with those without R6 when coinoculated with R6-virulent and avirulent isolates. R6 virulence in P. macularis appears to be race specific and, at this time, imposes a measurable fitness penalty on the fungus. Resistance genes R1 and R2 appear to remain effective against R6-virulent isolates of P. macularis in the U.S. Pacific Northwest.