Jerry E. Weiland

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.

Prediction of Long-Term Canker Disease Damage from the Responses of Juvenile Poplar Clones to Inoculation with Septoria musiva

Stem cankers caused by Septoria musiva severely limit production of susceptible hybrid poplars in eastern North America. A field experiment was conducted to determine whether short-term responses of poplar stems to inoculation with S. musiva were predictive of long-term canker disease damage. Stems of 27 poplar clones were inoculated during their first season of growth by removing the fourth or fifth fully expanded leaf and placing an agar plug colonized by an aggressive isolate of S. musiva over the resulting wound. Four months after inoculation, incidence of cankers, canker length, and percent of stem circumference affected (girdle) were recorded. Clones varied greatly in canker incidence (12 to 98%), mean canker length (10 to 53 mm), and mean girdle (14 to 94%). Logistic regression analysis was used to compare these inoculation responses with canker disease damage categories assigned on the basis of information from longer-term field studies. Incidence, canker length, and girdle data were all informative, but girdle data from this field experiment correctly predicted assigned canker disease damage categories most frequently (24 of 27 clones). Responses of 15 of these clones also were evaluated in a similar greenhouse experiment. Although responses usually were predictive of long-term damage categories, the probability of correct prediction was lower than that obtained in the field experiment for most clones. These results demonstrate the feasibility and potential benefit of screening juvenile poplar clones for responses to inoculation with S. musiva before undertaking field trials for productivity and subsequent release to growers.

Aggressiveness of Phytophthora cactorum, P. citricola I, and P. plurivora from European beech.

Phytophthora cactorum, P. citricola I, and P. plurivora cause bleeding cankers on mature European beech (Fagus sylvatica) trees in the northeastern United States. Inoculation experiments were conducted to compare the aggressiveness of the three Phytophthora spp. on stems, leaf disks, and roots of European beech and common lilac (Syringa vulgaris) seedlings. Isolates were obtained from bleeding cankers on European beech from five cities in New York (Albany, Ithaca, Oyster Bay, Plainview, and Rochester) and from a bleeding canker on sugar maple in Ithaca, NY. Stems were inoculated with colonized agar plugs, leaf disks with a zoospore suspension, and roots via infested soil at three inoculum levels. All organs of inoculated beech and lilac developed disease except for lilac roots inoculated with zoospores of P. cactorum. Disease incidence, severity, and plant survival were dependent on isolate and were also influenced by the tissue inoculated and host. Isolates of P. cactorum were the least aggressive and caused less necrosis than isolates of P. citricola I and P. plurivora. Results emphasize the utility of stem and root inoculation for evaluation of this canker disease and underscore critical differences in species aggressiveness.

Activity of Meadowfoam (Limnanthes alba) Seed Meal Glucolimnanthin Degradation Products against Soilborne Pathogens

Meadowfoam (Limnanthes alba L.) is a herbaceous winter-spring annual grown as a commercial oilseed crop. The meal remaining after oil extraction from the seed contains up to 4% of the glucosinolate glucolimnanthin. Degradation of glucolimnanthin yields toxic breakdown products, and therefore the meal may have potential in the management of soilborne pathogens. To maximize the pest-suppressive potential of meadowfoam seed meal, it would be beneficial to know the toxicity of individual glucolimnanthin degradation products against specific soilborne pathogens. Meloidogyne hapla second-stage juveniles (J2) and Pythium irregulare and Verticillium dahliae mycelial cultures were exposed to glucolimnanthin as well as its degradation products. Glucolimnanthin and its degradation product, 2-(3-methoxyphenyl)acetamide, were not toxic to any of the soilborne pathogens at concentrations up to 1.0 mg/mL. Two other degradation products, 2-(3-methoxymethyl)ethanethioamide and 3-methoxyphenylacetonitrile, were toxic to M. hapla and P. irregulare but not V. dahliae. The predominant enzyme degradation product, 3-methoxybenzyl isothiocyanate, was the most toxic compound against all of the soilborne pathogens, with M. hapla being the most sensitive with EC(50) values (0.0025 ± 0.0001 to 0.0027 ± 0.0001 mg/mL) 20-40 times lower than estimated EC(50) mortality values generated for P. irregulare and V. dahliae (0.05 and 0.1 mg/mL, respectively). The potential exists to manipulate meadowfoam seed meal to promote the production of specific degradation products. The conversion of glucolimnanthin into its corresponding isothiocyanate should optimize the biopesticidal properties of meadowfoam seed meal against M. hapla, P. irregulare, and V. dahliae.

The Histology of Hybrid Poplar Clones Inoculated with Septoria musiva

Septoria musiva causes stem cankers that severely limit production of susceptible hybrid poplars in eastern North America. A field experiment was conducted with resistant clone DN34 and susceptible clone NC11505 in order to (i) identify tissues colonized by the pathogen, (ii) describe tissue responses to S. musiva, and (iii) determine whether tissue responses to S. musiva differed between hybrid poplar clones. Branches of each clone were inoculated by removing the fourth or fifth fully expanded leaf and placing an agar plug colonized by an aggressive isolate of S. musiva over the wound. Seven weeks after inoculation, branches were harvested and prepared for histology. Data from nonwounded control, wounded control, and wounded and inoculated stems were collected and analyzed for effects of clone and treatment. In general, fungal colonization was more extensive in NC11505 and exophylactic and necrophylactic periderms (NPs) of clone DN34 were significantly thicker than those of NC11505, regardless of treatment. The number of NPs produced and the distance from the epidermis to the outermost layer of phellem were significantly affected by the pathogen. Inoculated stems of clone DN34 developed a single NP that formed closer to the wound surface than in wounded controls. In contrast, inoculated stems of NC11505 developed successive NPs and the first NP formed further from the wound surface than in wounded controls. These two host responses to inoculation, as well as measures of exophylactic and necrophylactic periderm thickness, may be useful as markers for the selection of poplar resistant to damage by S. musiva.

Effects of Mefenoxam, Phosphonate, and Paclobutrazol on In Vitro Characteristics of Phytophthora cactorum and P. citricola and on Canker Size of European Beech

Phytophthora citricola and P. cactorum cause bleeding cankers that lead to the death of mature European beech (Fagus sylvatica) in the northeastern United States. The effects of two fungicides and a plant growth regulator on in vitro pathogen characteristics and on canker expansion were investigated. In the first experiment, 16 single-spore isolates (11 P. citricola and 5 P. cactorum) were grown on clarified V8 juice agar amended with (i) 0 to 20 μg a.i./ml of mefenoxam, (ii) 0 to 301,429 μg a.i./ml phosphonate either with or without a bark-penetrating surfactant at 0.5 mg a.i./ml, or (iii) 0 to 25 mg a.i./ml of the surfactant alone. Radial growth, oospore production, and zoospore germination were observed to be dependent on isolate and treatment. A species effect on growth was also observed, as P. cactorum isolates were 2.5- to sevenfold less sensitive to phosphonate, but 2- to 150-fold more sensitive to mefenoxam than P. citricola isolates (based on 50% inhibition of growth). In the second experiment, bark and soil drenches of mefenoxam (50 mg a.i./ml and 19 μg a.i./ml, respectively), phosphonate (301,429 and 101 μg a.i./ml, respectively), and a soil drench of paclobutrazol (21 mg a.i./ml) were evaluated for their efficacy as curative or preventive treatments against bleeding canker. None of the treatments (curative or preventive) were able to stop canker expansion or prevent infection. However, saplings inoculated with P. citricola and treated with the phosphonate bark drench as either a curative or preventive treatment had cankers that were 36 to 82% shorter than those of inoculated control stems treated with water. For saplings inoculated with P. cactorum, the phosphonate bark drench was only effective when applied as a preventive (38% shorter than inoculated control stems treated with water), and not as a curative treatment. No other treatment was effective at limiting canker expansion.

Pythium Species and Isolate Diversity Influence Inhibition by the Biological Control Agent Streptomyces lydicus

Disease control of soilborne pathogens by biological control agents (BCAs) is often inconsistent under field conditions. This inconsistency may be partly influenced by pathogen diversity if there is a differential response among pathogen species and isolates to selected BCAs. The responses of 148 Pythium isolates obtained from soil at three forest nurseries and representative of 16 Pythium spp. were evaluated in the presence of Streptomyces lydicus strain WYEC108 in an in vitro assay. Percent growth inhibition, inhibition zone distance, mortality, and growth rate were recorded for each isolate, and data were analyzed for effects of species and isolate. Responses of three Pythium spp. (Pythium irregulare, P. sylvaticum, and P. ultimum) were further analyzed for a location (nursery) effect. Although S. lydicus inhibited all Pythium isolates, differences in percent growth inhibition, inhibition zone distance, and mortality were observed among Pythium spp. and isolates. Small but significant location effects were also noted. Growth rate also varied among Pythium spp. and isolates and was found to strongly bias percent growth inhibition and, to a lesser degree, inhibition zone distance; depending on which measure was used, slower-growing isolates appeared less sensitive (growth inhibition) or more sensitive (inhibition zone) to S. lydicus than faster-growing isolates. Results illustrate the importance of using multiple, representative pathogen isolates in preliminary BCA inhibition assays as well as accounting for the effect of pathogen growth rate on pathogen inhibition by BCAs. Future studies should take pathogen diversity into account when evaluating biological control efficacy.

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.

Identifying Rates of Meadowfoam (Limnanthes alba) Seed Meal Needed for Suppression of Meloidogyne hapla and Pythium irregulare in Soil

Meadowfoam (Limnanthes alba) is a commercial oilseed annual crop grown in Oregon. After extracting oil from seed, the remaining seed meal is rich in the secondary plant metabolite glucolimnanthin, which can be converted into pesticidal compounds such as 3-methoxybenzyl isothiocyanate (ITC) and 3-methoxyphenylacetonitrile (nitrile) in the presence of the enzyme myrosinase. In previous studies, we demonstrated that ITC and nitrile, produced by mixing freshly ground meadowfoam seed with meadowfoam seed meal, are toxic to the plant-parasitic nematode Meloidogyne hapla and the plant pathogen Pythium irregulare. In this study, we evaluated factors that might influence the implementation of meadowfoam seed meal into agricultural production systems for soilborne pathogen and nematode control. Rate-finding experiments demonstrated that a minimum 1.0% seed/seed meal formulation (wt/wt) was necessary to achieve nematode and pathogen suppression; seed meal alone was insufficient for complete control of M. hapla and stimulated the growth of P. irregulare. When this 1.0% seed/seed meal formulation was used, a greater soil amendment rate was required to cause 100% mortality of P. irregulare (1.0% wt/wt) than for M. hapla (0.5% wt/wt). In phytotoxicity experiments, soil amended with the 1.0% seed/seed meal formulation was consistently phytotoxic to wheat, cucumber, and tomato. However, phytotoxic effects were mitigated by a delayed planting into the amended soil. A final assay to monitor concentrations of ITC and nitrile in conjunction with assessing M. hapla and P. irregulare mortality was conducted over a 6-day period in soils amended at 0.5 and 1.0% (wt/wt) with the 1.0% seed/seed meal formulation. The response was rapid, with 100% mortality of both organisms within 2 h after exposure to amended soil. Concentrations of nitrile remained relatively constant over the 6-day period (approximately 0.017 and 0.032 mg/ml at 0.5 and 1.0% amendment rates, respectively), whereas ITC production increased rapidly and peaked 12 to 24 h after amendment (0.083 and 0.171 mg/ml at 0.5 and 1.0% amendment rates, respectively) before returning to near undetectable levels.

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