Fungicide Sensitivity of Phytophthora Isolates from the Washington Red Raspberry Industry.

Phytophthora rubi is an important pathogen causing Phytophthora root rot of red raspberries worldwide. Management of this disease is partially achieved with fungicides, but efficacy has been low, and growers are concerned about fungicide resistance. To determine whether fungicide resistance is developing, Phytophthora species were isolated from 26 raspberry fields with root rot, identified, and evaluated for sensitivity to four fungicides: mefenoxam, phosphorous acid, oxathiapiprolin, and dimethomorph. The majority of the recovered 152 Phytophthora isolates were P. rubi (143 isolates, 25 fields), with P. megasperma (8 isolates, 2 fields) and P. gonapodyides (1isolate, 1field) being found much less frequently. These results confirm P. rubi as the dominant species affecting the Washington red raspberry industry. Almost all tested isolates were sensitive to all four fungicide chemistries, although three isolates were less sensitive to mefenoxam, with effective concentration for 50% growth inhibition (EC50) values ranging from 3.53 to 100 µg active ingredient/ml. No resistance was detected against current fungicide label rates. However, other reasons were identified for why fungicides have been ineffective. Label rates vary widely by brand, and most fungicides are applied in the fall when P. rubi is inactive. In addition, some phosphorous acid products are only labeled for foliar applications, which have been shown to be less effective than soil applications in other agricultural systems. Efficacy trials are needed to compare foliar and soil fungicide applications at different times of the year for their ability to control Phytophthora root rot in red raspberry production fields.

The Effect of Boxwood Leaf Volatiles on Conidial Germination of Calonectria pseudonaviculata, the Causal Agent of Boxwood Blight.

The fungal pathogen Calonectria pseudonaviculata causes boxwood blight and is a significant threat to the boxwood industry, as well as historic boxwood gardens. The pathogen produces conidia in sticky masses that are splash dispersed, which germinate and infect through stomata on the leaves or stems, causing leaf spots and stem lesions. Despite its ability to cause severe infections on boxwood plants, the pathogen often has a low germination rate on artificial media under lab conditions. To identify cues that stimulate germination, we explored whether host factors could induce high germination rates. In this study, we demonstrate that C. pseudonaviculata spores achieve high germination rates when they are placed on detached leaves of boxwood and other known hosts, compared to potato dextrose agar and glass coverslips. We also demonstrate that germination is induced by volatiles from detached leaves of boxwood, as well as the nonhost Berberis thunbergii. When C. pseudonaviculata spores were exposed to volatiles from boxwood leaves in the presence of ethylene scrubber packs that contained potassium permanganate, the stimulatory effect on spore germination was reduced. However, ethylene, a regulator of leaf senescence, did not stimulate germination of C. pseudonaviculata spores. This suggests that the pathogen may have evolved to recognize one or more host volatiles, other than ethylene to induce germination, thus limiting its growth until it senses the presence of a host plant.

Temperature and Fungicide Sensitivity in Three Prevalent Phytophthora Species Causing Phytophthora Root Rot in Rhododendron.

Temperature is an important environmental variable affecting Phytophthora spp. biology. It alters the ability of species to grow, sporulate, and infect their plant host, and it is also important in mediating pathogen responses to disease control measures. Average global temperatures are increasing as a consequence of climate change, yet there are few studies that compare the effects of temperature on Phytophthora spp. that are important to the nursery industry. To address this, we conducted a series of experiments to evaluate how temperature affects the biology and control of three soilborne Phytophthora spp. prevalent in the nursery industry. In the first set of experiments, we evaluated the mycelial growth and sporulation of several Phytophthora cinnamomi, P. plurivora, and P. pini isolates at temperatures ranging from 4 to 42°C for different amounts of time (0 to 120 h). In the second set of experiments, we evaluated the response of three isolates of each species to the fungicides mefenoxam and phosphorous acid at temperatures ranging from 6 to 40°C. Results showed that each species responds differently to temperature, with P. plurivora having the greatest optimal temperature (26.6°C), P. pini the least (24.4°C), and P. cinnamomi was intermediate between the two (25.3°C). P. plurivora and P. pini had the lowest minimum temperatures (approximately 2.4°C) compared with P. cinnamomi (6.5°C), while all three species had a similar maximum temperature (approximately 35°C). When tested against mefenoxam, all three species were generally more sensitive to mefenoxam at cool temperatures (6 to 14°C) than at warmer temperatures (22 to 30°C). P. cinnamomi was also more sensitive to phosphorous acid at cool temperatures (6 to 14°C). However, both P. plurivora and P. pini tended to be more sensitive to phosphorous acid at warmer temperatures (22 to 30°C). These findings help define the temperatures at which these pathogens will be the most damaging and help delineate the temperatures at which fungicides should be applied for maximum efficacy.

Cool Temperatures Favor Growth of Oregon Isolates of Calonectria pseudonaviculata and Increase Severity of Boxwood Blight on Two Buxus Cultivars.

Controlled environment experiments were conducted to evaluate the effects of temperature on Calonectria pseudonaviculata mycelial growth and the effects of temperature and infection period on boxwood blight severity. In experiment 1, 15 Oregon isolates (representing five genotypes) were grown on potato dextrose agar (PDA) and malt extract agar (MEA) at six temperatures from 5 to 30°C. Growth (culture diameter) was measured after 2 weeks. Optimal growth occurred at 25°C on PDA and 20°C on MEA. Isolates of genotype G1 also grew faster than genotype G2, but only on MEA at 25°C. In experiment 2, Buxus cultivars Green Velvet (GV, more susceptible) and Winter Gem (WG, more resistant) were inoculated and incubated in moist chambers for 9 or 24 h at 22°C (infection period), then moved into growth chambers at 15 or 25°C. After 4 weeks, chamber temperatures were switched, and plants were incubated for 4 more weeks. Disease severity was evaluated weekly. During the first 4 weeks, disease was generally more severe on GV than WG, on plants with a 24-h versus a 9-h infection period, and on plants incubated at 15°C versus 25°C. However, disease was just as severe on WG as GV when the 24-h infection period was followed by incubation at 15°C. After the temperatures were switched, disease increased only on WG that were cooled from 25 to 15°C. Results show that Oregon isolates of C. pseudonaviculata are capable of growing faster and causing more severe disease at temperatures cooler than those reported previously.

Is Disease Induced by Flooding Representative of Nursery Conditions in Rhododendrons Infected with Phytophthora cinnamomi or P. plurivora?

The degree of flooding commonly used to induce disease in Phytophthora root rot studies rarely occurs in container nurseries. Instead, over-irrigation and poor drainage result in plants periodically sitting in shallow pools of water. Rhododendron plants were grown in a noninfested substrate or substrate infested with Phytophthora cinnamomi or P. plurivora to determine whether root rot induced by flooding represents disease that occurs under simulated nursery conditions when plants are in a shallow pool of water (saucers), or are allowed to freely drain and maintained at ∼75% container capacity (CC). Generally P. cinnamomi caused more disease than P. plurivora, and all water treatments were conducive to root rot. In experiment 1, the amount of disease caused by flooding was similar to that in the saucer treatment (75% CC not tested) while in experiment 2, flooding often caused more rapid and severe disease than the saucer or 75% CC treatment. Pathogens differed in their response to water treatments. P. cinnamomi caused more disease in treatments with >90% substrate moisture for either a short (flood) or long duration (saucer), while P. plurivora was less capable of causing disease when soil moisture was maintained >90% than when substrate moisture was maintained at a more moderate level (flood, 75% CC). Our results indicate that it is not necessary to flood plants to induce disease under experimental conditions and that disease induced by flooding can represent disease in container nurseries when containers are in pools of water or maintained at ∼75% CC. In addition, our results suggest that P. cinnamomi is a more aggressive pathogen than P. plurivora in nursery conditions where drainage is poor; however, both species are capable of causing a similar amount of disease under more typical irrigation management.

Virulence of Five Phytophthora Species Causing Rhododendron Root Rot in Oregon.

Phytophthora root rot is a destructive disease of rhododendron that causes substantial losses of this nursery crop in infested field and container production areas. Historically, Phytophthora cinnamomi was considered the main causal agent of the disease. However, a recent survey of soilborne Phytophthora species from symptomatic rhododendrons in Oregon revealed that P. plurivora is more common than P. cinnamomi, and that several other Phytophthora species may be involved. We investigated the ability of the five most abundant species from the survey to cause root rot: P. plurivora, P. cinnamomi, P. pini, P. pseudocryptogea, and P. cambivora. Three to four isolates were selected for each species from across six Oregon nurseries. Media of containerized Rhododendron catawbiense 'Boursault' was infested with single isolates in a randomized complete block design in a greenhouse. Phytophthora cinnamomi, P. pini, and P. plurivora rapidly caused ≥90% of severe root rot, whereas P. pseudocryptogea caused more moderate disease (46% of severe root rot). Phytophthora cambivora failed to produce enough inoculum and was used at a lower inoculum density than the other four species; however, occasionally, it caused severe root rot (5% incidence). No differences in virulence were observed among isolates of the same species, except for one isolate of P. plurivora that caused less disease than other P. plurivora isolates. This study demonstrates that all five Phytophthora species, which were representative of 94% of the survey isolates, are capable of causing severe root rot and plant death, but that not all species are equally virulent.

Phytophthora Species Differ in Response to Phosphorous Acid and Mefenoxam for the Management of Phytophthora Root Rot in Rhododendron.

Phytophthora root rot, caused by many soilborne Phytophthora spp., is a significant disease affecting the $42 million rhododendron nursery industry. Rhododendron growers have increasingly reported failure by two systemic fungicides, phosphorous acid and mefenoxam, to adequately control root rot. Both fungicides may be applied as a foliar spray or soil drench but it is unknown how application method, fungicide chemistry, or pathogen diversity affects disease control. Therefore, two experiments were conducted to (i) determine whether differences in application method or fungicide chemistry affect control of root rot caused by P. cinnamomi and P. plurivora and (ii) evaluate the sensitivity of Phytophthora spp. and isolates from the rhododendron industry to each fungicide. Results demonstrated that soil drenches of either fungicide were more effective than foliar sprays for control of P. cinnamomi but were ineffective for P. plurivora. Furthermore, Phytophthora spp. and isolates varied in sensitivity to phosphorous acid and mefenoxam, and there were multiple fungicide-insensitive isolates, especially within P. plurivora. Differences in sensitivity were also observed among isolates from different nurseries and production systems, with some nurseries having less sensitive isolates than others and with container systems generally having less sensitive isolates than field systems. Our results provide three potential reasons for why fungicide control of Phytophthora root rot might fail: (i) the fungicide can be applied to the wrong portion of the plant for optimal control, (ii) there are differences in fungicide sensitivity among soilborne Phytophthora spp. and isolates infecting rhododendron, and (iii) fungicide-insensitive isolates are present in the rhododendron nursery industry.

Growth, Sporulation, and Pathogenicity of the Raspberry Pathogen Phytophthora rubi Under Different Temperature and Moisture Regimes.

Phytophthora root rot of raspberry, which is mostly caused by Phytophthora rubi, is a significant issue for the Washington State red raspberry industry. Considered a cool weather pathogen, it is often assumed that it is most active and infective during the cool, wet winters of the region when soil temperatures range from 5 to 10°C; however, there are little data to support this view. More recent research has found that symptoms of root disease during late summer were strongly associated with P. rubi. Therefore, experiments were conducted at four temperatures from 5 to 20°C to evaluate the effects of temperature on P. rubi mycelial growth and sporulation and the effects of both temperature and soil moisture on the pathogenicity of P. rubi on red raspberry. At 20°C, P. rubi grew fastest and sporulated the most heavily. However, disease was most severe at both 15 and 20°C. The soil moisture parameters tested did not affect the pathogenicity results. These results show that P. rubi is more likely to infect during the spring and summer months (from May through September), when soil temperatures are consistently in the range of 15 to 20°C.

Soilborne Phytophthora and Pythium Diversity From Rhododendron in Propagation, Container, and Field Production Systems of the Pacific Northwest.

Rhododendron root rot is a severe disease that causes significant mortality in rhododendrons. Information is needed about the incidence and identity of soilborne Phytophthora and Pythium species causing root rot in Pacific Northwest nurseries in order to better understand the disease etiology and to optimize disease control strategies. The last survey focusing solely on soilborne oomycete pathogens in rhododendron production was conducted in 1974. Since then, advances in pathogen identification have occurred, new species may have been introduced, pathogen communities may have shifted, and little is known about Pythium species affecting this crop. Therefore, a survey of root-infecting Phytophthora and Pythium species was conducted at seven nurseries from 2013 to 2017 to (i) document the incidence of root rot damage at each nursery and stage of production, (ii) identify soilborne oomycetes infecting rhododendron, and (iii) determine whether there are differences in pathogen diversity among nurseries and production systems. Rhododendrons from propagation, container, and field systems were sampled and Phytophthora and Pythium species were isolated from the roots and collar region. Root rot was rarely evident in propagation systems, which were dominated by Pythium species. However, severe root rot was much more common in container and field systems where the genus Phytophthora was also more prevalent, suggesting that Phytophthora species are the primary cause of severe root rot and that most contamination by these pathogens comes in after the propagation stage. In total, 20 Pythium species and 11 Phytophthora species were identified. Pythium cryptoirregulare, Pythium aff. macrosporum, Phytophthora plurivora, and Phytophthora cinnamomi were the most frequently isolated species and the results showed that Phytophthora plurivora has become much more common than in the past. Phytophthora diversity was also greater in field systems than in propagation or container systems. Risks for Phytophthora contamination were commonly observed during the survey and included placement of potting media in direct contact with field soil, the presence of dead plants that could serve as continuous sources of inoculum, and the presence of excess water as a result of poor drainage, overirrigation, or malfunctioning irrigation equipment. In the past, research on disease development and root rot disease control in rhododendron focused almost exclusively on Phytophthora cinnamomi. More research is needed on both of these topics for the other root-infecting species identified in this survey.

Variation in Disease Severity Caused by Phytophthora cinnamomi, P. plurivora, and Pythium cryptoirregulare on Two Rhododendron Cultivars.

Rhododendrons are an important crop in the ornamental nursery industry, but are prone to Phytophthora root rot. Phytophthora root rot is a continuing issue on rhododendrons despite decades of research. Several Phytophthora species are known to cause root rot, but most research has focused on P. cinnamomi, and comparative information on pathogenicity is limited for other commonly encountered oomycetes, including Phytophthora plurivora and Pythium cryptoirregulare. In this study, three isolates each of P. cinnamomi, P. plurivora, and Py. cryptoirregulare were used to inoculate rhododendron cultivars Cunningham's White and Yaku Princess at two different inoculum levels. All three species caused disease, especially at the higher inoculum level. P. cinnamomi and P. plurivora were the most aggressive pathogens, causing severe root rot, whereas Py. cryptoirregulare was a weak pathogen that only caused mild disease. Within each pathogen species, isolate had no influence on disease. Both P. cinnamomi and P. plurivora caused more severe disease on Cunningham's White than on Yaku Princess, suggesting that the relative resistance and susceptibility among rhododendron cultivars might be similar for both pathogens. Reisolation of P. cinnamomi and P. plurivora was also greater from plants exhibiting aboveground symptoms of wilting and plant death and belowground symptoms of root rot than from those without symptoms. Results show that both P. cinnamomi and P. plurivora, but not Py. cryptoirregulare, are important pathogens causing severe root rot in rhododendron. This study establishes the risks for disease resulting from low and high levels of inoculum for each pathogen. Further research is needed to evaluate longer term risks associated with low inoculum levels on rhododendron health and to explore whether differences among pathogen species affect disease control.

Late-summer Disease Symptoms in Western Washington Red Raspberry Fields Associated with Co-Occurrence of Phytophthora rubi, Verticillium dahliae, and Pratylenchus penetrans, but not Raspberry bushy dwarf virus.

Sixty percent of the $109 million processed red raspberry industry of the United States occurs in northern Washington State. In 2012, late-summer symptoms of vascular wilt and root disease were observed in many raspberry plantings. These symptoms were initially attributed to Verticillium dahliae. However, diagnostic tests for the pathogen were often contradictory and other soilborne pathogens (Phytophthora rubi and Pratylenchus penetrans) or Raspberry bushy dwarf virus (RBDV) might also have been involved. Therefore, a survey was conducted in 2013 and 2014 to (i) establish the incidence and soil population levels of V. dahliae in red raspberry production fields, (ii) compare among diagnostic methods and laboratories for detecting and quantifying V. dahliae from raspberry field soil, and (iii) assess which pathogens are associated with late-summer disease symptoms of raspberry. Plant and soil samples were collected from 51 disease sites and 20 healthy sites located in 24 production fields. Samples were analyzed for the presence and quantity of each pathogen using traditional plating and extraction methods (V. dahliae, P. rubi, and P. penetrans), quantitative polymerase chain reaction (qPCR) (V. dahliae and P. rubi), and enzyme-linked immunosorbent assay (RBDV). Results showed that V. dahliae was present in 88% of the production fields and that detection of the pathogen differed by method and by laboratory: qPCR detected V. dahliae in the soil from approximately three times as many sites (51 of 71 total sites) as by plating on NP10 semi-selective medium (15 of 71 total sites). Soil populations of V. dahliae were slightly greater at disease sites, but the pathogen was detected with similar frequency from healthy sites and it was rarely isolated from diseased plants (4%). P. rubi, P. penetrans, and RBDV were also common in production fields (79, 91, and 53% of fields, respectively). Both P. rubi (soil and root samples) and P. penetrans (root populations only), but not RBDV, were more frequently found at disease sites than healthy sites, and the amount of P. rubi detected by qPCR was greater from disease sites than healthy sites. In addition, P. rubi was isolated from 27% of the symptomatic plants located at disease sites. Regardless of detection method, V. dahliae, P. rubi, and P. penetrans, either with or without RBDV, were more likely to co-occur at disease sites (73%) than healthy sites (35%), suggesting that a soilborne disease complex is present in raspberry production fields. Results indicate that P. rubi is the primary pathogen most strongly associated with late-summer symptoms of disease, but root populations of P. penetrans and higher soil populations of V. dahliae may also be of concern. Therefore, disease control methods should focus on all three soilborne pathogens.

Pathogenicity and Virulence of Pythium Species Obtained from Forest Nursery Soils on Douglas-Fir Seedlings.

Pythium species are common soilborne oomycetes that occur in forest nursery soils throughout the United States. Numerous species have been described from nursery soils. However, with the exception of P. aphanidermatum, P. irregulare, P. sylvaticum, and P. ultimum, little is known about the potential for other Pythium species found in nursery soils to cause damping-off of tree seedlings. A greenhouse study was conducted to evaluate the pathogenicity and virulence of 44 Pythium isolates representing 16 species that were originally recovered from soil at three forest nurseries in Washington and Oregon. Seeds of Douglas-fir (Pseudotsuga menziesii) were planted into soil infested with each of the isolates. Seedling survival, the number of surviving seedlings with necrotic root lesions, and taproot length were evaluated 4 weeks later. Responses of Douglas-fir to inoculation varied significantly depending on Pythium species and isolate. Eight species (P. dissotocum, P. irregulare, P. aff. macrosporum, P. mamillatum, P. aff. oopapillum, P. rostratifingens, P. sylvaticum, and P. ultimum var. ultimum) significantly reduced the number of surviving seedlings compared to the noninoculated treatment. However, all Pythium species caused a greater percentage of seedlings to develop root lesions (total mean 40%) than was observed from noninoculated seedlings (17%). Taproot length varied little among Pythium treatments and was not a useful character for evaluating pathogenicity. Results confirm the ability of P. irregulare, P. mamillatum, and P. ultimum var. ultimum to cause damping-off of Douglas-fir seedlings, and are indicative that other species such as P. dissotocum, P. aff. macrosporum, P. aff. oopapillum, P. rostratifingens, and P. sylvaticum may also be responsible for seedling loss.