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.

Rapid and Scalable DNA Extraction and Real-Time PCR Assay from Boxwood Tissue for the Detection of Calonectria pseudonaviculata, Causal Agent of Boxwood Blight.

Boxwood blight can be challenging to detect in the field, especially when symptoms are mild, thus requiring large numbers of plants to be screened. Therefore, a rapid diagnostic assay that can detect the pathogen from large amounts of plant tissue would be useful. Here, we present a crude DNA extraction protocol that is rapid and scalable. The DNA extraction protocol can process large volumes of tough boxwood tissue rapidly without using cetyltrimethylammonium bromide or phenol-chloroform to remove inhibitors. Additionally, to detect the boxwood blight pathogen Calonectria pseudonaviculata, we developed a TaqMan probe to use with previously described PCR primers for a real-time PCR assay. The assay's limit of detection was determined by diluting symptomatic boxwood leaves in nondiseased leaves and by adding spores to nondiseased leaves to simulate diagnostic scenarios. The assay was able to detect the pathogen in symptomatic leaves diluted up to 1 × 104- to 1 × 105-fold in nondiseased leaves and from as low as 1,000 to 10,000 spores added to 1.2 g of nondiseased leaves. The ability to extract DNA from large volumes of plant tissue facilitates screening more plant tissue using the real-time PCR assay without increasing the number of samples to process in the lab.

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.

Phylogeography of the wide-host range panglobal plant pathogen Phytophthora cinnamomi.

Various hypotheses have been proposed regarding the origin of the plant pathogen Phytophthora cinnamomi. P. cinnamomi is a devastating, highly invasive soilborne pathogen associated with epidemics of agricultural, horticultural and forest plantations and native ecosystems worldwide. We conducted a phylogeographic analysis of populations of this pathogen sampled in Asia, Australia, Europe, southern and northern Africa, South America, and North America. Based on genotyping-by-sequencing, we observed the highest genotypic diversity in Taiwan and Vietnam, followed by Australia and South Africa. Mating type ratios were in equal proportions in Asia as expected for a sexual population. Simulations based on the index of association suggest a partially sexual, semi-clonal mode of reproduction for the Taiwanese and Vietnamese populations while populations outside of Asia are clonal. Ancestral area reconstruction provides new evidence supporting Taiwan as the ancestral area, given our sample, indicating that this region might be near or at the centre of origin for this pathogen as speculated previously. The Australian and South African populations appear to be a secondary centre of diversity following migration from Taiwan or Vietnam. Our work also identified two panglobal, clonal lineages PcG1-A2 and PcG2-A2 of A2 mating type found on all continents. Further surveys of natural forests across Southeast Asia are needed to definitively locate the actual centre of origin of this important plant pathogen.

Optimizing Inoculum Production Methods for Infesting Soil with Phytophthora Species.

Inoculum production is an important part of conducting research with soilborne Phytophthora species. One common method is to incubate Phytophthora cultures in nutrient-amended vermiculite. However, inoculum levels often vary among batches of inoculum, even when production methods remain the same, and incubation typically takes ≥6 weeks, increasing risks for delayed experiments if the resulting inoculum level is too low. A more reliable and rapid method is needed for future studies. Experiments were conducted to determine inoculum levels of Phytophthora cinnamomi and Phytophthora plurivora after incubation in V8 juice-amended vermiculite (standard method); evaluate how inoculum viability was affected by air-drying; develop a modified method that takes less time to produce a vermiculite-based inoculum; and evaluate the effect of storage on inoculum viability. Results showed that the standard method produced inoculum levels from 716 to 1,808 colony forming units (CFUs)/g and that drying to <78% moisture content significantly reduced viability. The modified method used 2-week-old Phytophthora cultures to infest vermiculite at 80% moisture content and produced inoculum levels from 214 to 525 CFU/g. Storage for >1 day generally reduced inoculum viability. Although inoculum levels from the modified method were lower than the standard method, inoculum levels for each isolate were more consistent between trials, and the modified method was 6 to 8 weeks faster. Production with the modified method can also be easily scaled up by infesting a greater volume of vermiculite with additional cultures of Phytophthora. These results are important because they help explain variability in soilborne Phytophthora inoculum production and storage and provide a new method for producing inoculum more quickly.

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.

One Clonal Lineage of Calonectria pseudonaviculata Is Primarily Responsible for the Boxwood Blight Epidemic in the United States.

Boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae is destroying cultivated and native boxwood worldwide, with profound negative economic impacts on the horticulture industry. First documented in the United States in 2011, the disease has now occurred in 30 states. Previous research showed that global C. pseudonaviculata populations prior to 2014 had a clonal structure, and only the MAT1-2 idiomorph was observed. In this study, we examined C. pseudonaviculata genetic diversity and population structure in the United States after 2014, following the expansion of the disease across the country over the past 5 years. Two hundred eighteen isolates from 21 states were genotyped by sequencing 11 simple sequence repeat (SSR) loci and by MAT1 idiomorph typing. All isolates presented C. pseudonaviculata-specific alleles, indicating that C. henricotiae is still absent in the U.S. states sampled. The presence of only the MAT1-2 idiomorph and gametic linkage disequilibrium suggests the prevalence of asexual reproduction. The contemporary C. pseudonaviculata population is characterized by a clonal structure and composed of 13 multilocus genotypes (SSR-MLGs) unevenly distributed across the United States. These SSR-MLGs grouped into two clonal lineages (CLs). The predominant lineage CL2 (93% of isolates) is the primary contributor to U.S. disease expansion. The contemporary U.S. C. pseudonaviculata population is not geographically subdivided and not genetically differentiated from the U.S. population prior to 2014, but is significantly differentiated from the main European population, which is largely composed of CL1. Our findings provide insights into the boxwood blight epidemic that are critical for disease management and breeding of resistant boxwood cultivars.

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.

Directed energy system technology for the control of soilborne fungal pathogens and plant-parasitic nematodes.

BACKGROUND: It is challenging to manage soilborne pathogens and plant-parasitic nematodes using sustainable practices. Here, we evaluated a novel energy application system, Directed Energy System (DES). This system generates pulses of energy capable of impacting selected biological organisms. The oomycete Phytophthora cinnamomi, the fungus Verticillium dahliae, and the plant-parasitic nematodes Meloidogyne hapla and Globodera ellingtonae were added to soil. Then DES-generated energy was applied to soil and impacts on target organisms were determined. RESULTS: DES applied at 20, 40 and 70 J cm-3 to P. cinnamomi and V. dahliae resulted in ≥50% and 92% reductions (respectively) of propagules per gram of soil in comparison to the untreated control. There was a significant reduction of M. hapla eggs per gram of host tomato root between the untreated control, and 2.2, 13 and 25 J cm-3 DES dosages applied pre- or post-planting. Additionally, an 84% reduction in hatch from G. ellingtonae encysted eggs after treatment with 70 J cm-3 DES was observed. The dosages ranged from 40 or 80V mm-1 for nematodes to 200 V mm-1 for fungi. CONCLUSION: DES-generated energy reduced survival of the soilborne pathogens P. cinnamomi and V. dahlia, and the plant-parasitic nematodes M. hapla and G. ellingtonae. The application of this technology to a field setting remains to be considered. Published 2020. This article is a U.S. Government work and is in the public domain in the USA. Pest Management Science published by Wiley Periodicals, Inc. on behalf of © 2020 Society of Chemical Industry.

Population Structure of Phytophthora plurivora on Rhododendron in Oregon Nurseries.

Phytophthora plurivora is a recently described plant pathogen, formerly recognized as P. citricola. Recent sampling of Pacific Northwest nurseries frequently encountered this pathogen, and it has been shown to be among the most damaging Phytophthora pathogens on ornamentals. We characterized the population structure of P. plurivora in a survey of four Oregon nurseries across three different counties with focus on Rhododendron hosts. Isolates were identified to the species level by Sanger sequencing and/or a PCR-RFLP assay of the internal transcribed spacer (ITS) region. We used genotyping-by-sequencing to determine genetic diversity. Variants were called de novo, resulting in 284 high-quality variants for 61 isolates after stringent filtering. Based on Fst and AMOVA, populations were moderately differentiated among nurseries. Overall, population structure suggested presence of one dominant clonal lineage in all nurseries, as well as isolates of cryptic diversity mostly found in one nursery. Within the clonal lineage, there was a broad range of sensitivity to mefenoxam and phosphorous acid. Sensitivity of the two fungicides was correlated. P. plurivora was previously assumed to spread clonally, and the low genotypic diversity observed within and among isolates corroborated this hypothesis. The broad range of fungicide sensitivity within the P. plurivora population found in PNW nurseries has implications for managing disease caused by this important nursery pathogen. These findings provide the first perspective into P. plurivora population structure and phenotypic plasticity in Pacific Northwest nurseries.

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.

Distribution and Longevity of Pratylenchus penetrans in the Red Raspberry Production System.

One of the major constraints on the production of red raspberries in the Pacific Northwest is the presence of the root-lesion nematode Pratylenchus penetrans. Current management of this nematode relies heavily on preplant soil fumigation; however, regulations have made the practice more difficult and expensive. Additional issues with soil fumigation include lack of efficacy at deeper soil depths and potential inability to penetrate raspberry root material that remains in the field during fumigation which may harbor P. penetrans. To address these issues, two field experiments were conducted in northwestern Washington. In the first experiment, the residency time of P. penetrans in root material from the previous raspberry crop, which was terminated with or without the use of herbicides, was monitored over time. Pratylenchus penetrans was found in root material from 6 to 8 mon after the crop was terminated, and herbicide application did not reduce P. penetrans residency time compared to untreated root material. In a second experiment, the vertical distribution of P. penetrans at three different times during the field establishment process (pre- and postfumigation, and at planting) was determined at two locations. Both locations had detectable prefumigation P. penetrans populations at all depths. However, postfumigation populations showed a different distribution pattern between locations. The location with coarser soil had populations located mainly at shallower depths with a maximum of 44 P. penetrans/100 g soil at 16 to 30 cm deep, whereas the location with finer soil had populations located mainly at deeper depths with a maximum of 8 P. penetrans/100 g soil at 76 to 90 cm deep. At planting, distribution tended to equilibrate among depths at both locations, but the overall population pattern across depth at each location was similar to that observed at postfumigation. Understanding more about the residency time and distribution of this nematode may provide growers with information that can be used to more effectively target P. penetrans.

Population Structure of Pythium irregulare, P. ultimum, and P. sylvaticum in Forest Nursery Soils of Oregon and Washington.

Pythium species are important soilborne pathogens occurring in the forest nursery industry of the Pacific Northwest. However, little is known about their genetic diversity or population structure and it is suspected that isolates are moved among forest nurseries on seedling stock and shared field equipment. In order to address these concerns, a total of 115 isolates of three Pythium species (P. irregulare, P. sylvaticum, and P. ultimum) were examined at three forest nurseries using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Analyses revealed distinct patterns of intraspecific variation for the three species. P. sylvaticum exhibited the most diversity, followed by P. irregulare, while substantial clonality was found in P. ultimum. For both P. irregulare and P. sylvaticum, but not P. ultimum, there was evidence for significant variation among nurseries. However, all three species also exhibited at least two distinct lineages not associated with the nursery of origin. Finally, evidence was found that certain lineages and clonal genotypes, including fungicide-resistant isolates, are shared among nurseries, indicating that pathogen movement has occurred.