Transmission of Spinach Downy Mildew via Seed and Infested Leaf Debris.

Spinach downy mildew, caused by the obligate oomycete pathogen Peronospora effusa, is a worldwide constraint on spinach production. The role of airborne sporangia in the disease cycle of P. effusa is well established, but the role of the sexual oospores in the epidemiology of P. effusa is less clear and has been a major challenge to examine experimentally. To evaluate seed transmission of spinach downy mildew via oospores in this study, isolated glass chambers were employed in two independent experiments to grow out oospore-infested spinach seed and noninfested seeds mixed with oospore-infested crop debris. Downy mildew diseased spinach plants were observed 37 and 34 days after planting in the two isolator experiments, respectively, in the chambers that contained one of two oospore-infested seed lots or seeds coated with oospore-infested leaves. Spinach plants in isolated glass chambers initiated from seeds without oospores did not show downy mildew symptoms. Similar findings were obtained using the same seed lot samples in a third experiment conducted in a growth chamber. In direct grow out tests to examine oospore infection on seedlings performed in a containment greenhouse with oospore-infested seed of two different cultivars, characteristic Peronospora sporangiophores were observed growing from a seedling of each cultivar. The frequency of seedlings developing symptoms from 82 of these oospore-infested seed indicated that approximately 2.4% of seedlings from infested seed developed symptoms, and 0.55% of seedlings from total seeds assayed developed symptoms. The results provide evidence that oospores can serve as a source of inoculum for downy mildew and provide further evidence of direct seed transmission of the downy mildew pathogen to seedlings in spinach via seedborne oospores.

The Impatiens Downy Mildew Epidemic in the United States Is Caused by New, Introgressed Lineages of Plasmopara destructor with Prominent Genotypic Diversity and High Evolutionary Potential.

Impatiens downy mildew (IDM) caused by Plasmopara destructor is currently the primary constraint on the production and use of impatiens (Impatiens walleriana) as bedding plants worldwide. Downy mildew has been documented since the 1880s from wild-grown Impatiens spp. but epidemic outbreaks of the disease affecting the commercially grown, ornamental I. walleriana were only reported for the first time in 2003 in the United Kingdom and in 2004 in the United States. Here, we assess the genetic diversity, level of differentiation, and population structure from 623 samples associated with current and preepidemic IDM outbreaks, by genotyping the samples with simple sequence repeat markers. P. destructor population structure following the emergence of IDM in the United States is subdivided into four genetic lineages characterized by high genetic diversity, mixed reproduction mode, inbreeding, and an excess of heterozygosity. P. destructor genotypes are significantly differentiated from preepidemic IDM samples from hosts other than I. walleriana but no geographical or temporal subdivision is evident. P. destructor samples from different Impatiens spp. show significant but very low levels of differentiation in the analysis of molecular variance test that did not hold in discriminant analysis of principal components analyses. The same was observed between samples of P. destructor and P. velutina recovered from I. walleriana. The finding of shared genotypes in samples from different countries and lack of differentiation among U.S. and Costa Rican samples indicate the occurrence of international movement of the pathogen. Our study provides the first high-resolution analysis of the diversity of P. destructor populations and the IDM epidemic that may be instrumental for disease management and breeding efforts.

First Report of Powdery Mildew of American Ginseng Caused by Erysiphe heraclei in Tennessee and the United States.

American ginseng (Panax quinquefolius L.), native to the forested regions of northeast U.S is a perennial herb valued as traditional Chinese medicine. It has been cultivated in North America for several decades due to high global demand. Powdery mildew symptoms were observed on 8-year-old cultivated American ginseng leaves (Fig. 1a, b) on a residential property in Rutherford Co., TN in May 2022. Disease severity was 40 to 60% of leaf area and incidence was 33% out of 30 plants. Affected plants exhibited white fungal colonies on the leaves. Under severe infection, the leaves were chlorotic and senescing. Microscopic observation revealed masses of conidia and mycelia on symptomatic tissue. Conidiophores were cylindrical and unbranched (2- or, rarely, 3-septate), measuring 66.7 ± 12.5 µm (n=78) with a range of 24.3 to 90.7 µm. Conidia produced singly or in pseudo-chains (Fig. 1c). Conidiophore foot cells measured 23.2 ± 4.3 µm long (n=54) and the width at the foot cell septum was 5.1 ± 0.6 µm (n=54). Hyphal width was 3.3 ± 0.6 um (n=59). Fresh vacuolated spores were oblong-elliptical to oblong (Fig. 1d) and measured 31.5 × 11.9 µm (n=55), lacked fibrosin bodies. The length-to-width ratio of conidia was 1.9 to 4.4 (avg. 2.7). Superficial mycelia and germinating spores displayed lobed appressorium (Fig. 1e). Detached spore surfaces were wrinkled (Fig. 1f). Morphological characteristics of the fungus matched the description of Erysiphe heraclei (Braun and Cook, 2012) and Erysiphe sp. (Cho et al. 2016) except for conidiophore length, which was shorter in our sample. To confirm pathogen identity, total DNA was extracted directly from single spore cultures (isolates FBG1668 and FBG1728). The ribosomal internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers (White et al. 1990). The sequences (GenBank accession nos. OP458196 and OP469994) showed 100% identity and 100% query coverage to E. heraclei (KY073878 and LC270862). The sequences were also 100% identical to the ITS sequences of E. betae and E. malvae. Solano-Báez et al. (2022) noted that the species in the E. malvae/E. heraclei/E. betae species complex are phylogenetically undistinguishable. E. betae and E. malvae infect plants in Chenopodiaceae and Malvaceae, respectively (Braun and Cook, 2012). However, E. heraclei has been reported to infect plants in Apiaceae. American ginseng belongs to Araliaceae which is a close family to Apiaceae and both belong to Apiales. Based on morphological and molecular identification, both isolates were identified as E. heraclei. Pathogenicity was confirmed by inoculating the adaxial leaf surface of six 2-year-old American ginseng plants. Spores from detached symptomatic leaves were tapped onto the adaxial surface of healthy leaves. Six non-inoculated and inoculated plants were maintained in a greenhouse at 21 to 23°C, 70%RH, with 16-h photoperiod. After 2 weeks, powdery mildew symptoms developed on the inoculated plants. The microscopy and molecular analysis confirmed infection and all control plants remained asymptomatic. Cho et al. (2016) reported powdery mildew on Korean ginseng (P. ginseng C.A. Mey) caused by Erysiphe sp., and Sholberg et al. (1996) reported Erysiphe sp. on P. quinquefolius in Canada, but to our knowledge, this is the first report of powdery mildew caused by E. heraclei on American ginseng in Tennessee and the U.S. Identification and timely management of powdery mildew on American ginseng will be necessary to control this disease in affected growing sites.

Impact of Calonectria Diseases on Ornamental Horticulture: Diagnosis and Control Strategies.

Diseases caused by fungi in the genus Calonectria pose a significant threat to the ornamental horticulture industries in Europe and the United States. Calonectria spp. are particularly challenging pathogens to manage in ornamental production systems and the urban landscape for multiple reasons. A high level of species diversity and poorly resolved taxonomy in the genus makes proper pathogen identification and disease diagnosis a challenge, though recent molecular phylogenetic studies have made significant advances in species delimitation. From a disease management perspective, Calonectria spp. produce long-lived survival structures (microsclerotia) that contaminate nursery production systems and can survive multiple years in the absence of a susceptible plant host. Latent infection of plant material is poorly understood but likely contributes to long-distance dissemination of these fungal pathogens, including the clonal Calonectria spp. responsible for the global emergence of boxwood blight. Breeding for disease resistance represents a sustainable strategy for managing Calonectria diseases but is challenging due to the perennial nature of many ornamental plants and high levels of susceptibility in commercial cultivars. Ultimately, long-term sustainable management of Calonectria diseases will require an improved understanding of pathogen biology as well as integration of multiple disease management strategies.

Comparative analysis of extracellular proteomes reveals putative effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata.

Calonectria henricotiae (Che) and C. pseudonaviculata (Cps) are destructive fungal pathogens causing boxwood blight, a persistent threat to horticultural production, landscape industries, established gardens, and native ecosystems. Although extracellular proteins including effectors produced by fungal pathogens are known to play a fundamental role in pathogenesis, the composition of Che and Cps extracellular proteins has not been examined. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics prediction tools, 630 extracellular proteins and 251 cell membrane proteins of Che and Cps were identified in the classical secretion pathway in the present study. In the non-classical secretion pathway, 79 extracellular proteins were identified. The cohort of proteins belonged to 364 OrthoMCL clusters, with the majority (62%) present in both species, and a subset unique to Che (19%) and Cps (20%). These extracellular proteins were predicted to play important roles in cell structure, regulation, metabolism, and pathogenesis. A total of 124 proteins were identified as putative effectors. Many of them are orthologs of proteins with documented roles in suppressing host defense and facilitating infection processes in other pathosystems, such as SnodProt1-like proteins in the OrthoMCL cluster OG5_152723 and PhiA-like cell wall proteins in the cluster OG5_155754. This exploratory study provides a repository of secreted proteins and putative effectors that can provide insights into the virulence mechanisms of the boxwood blight pathogens.

First report: Co-infection of Sarcococca hookeriana (sweetbox) by Coccinonectria pachysandricola and Calonectria pseudonaviculata causes a foliar disease of sweetbox in Pennsylvania.

Sweetbox (Sarcococca hookeriana) are high value ornamental shrubs susceptible to disease caused by Calonectria pseudonaviculata (Cps) and Coccinonectria pachysandricola (Cpa) (Malapi-Wight et al. 2016; Salgado-Salazar et al. 2019). In July 2018, 18-month old sweetbox with leaf spots and defoliation were observed in a residential landscape in Lancaster County, Pennsylvania. Small tan leaf spots grew to cover half of the leaf, developing a concentric banding with dark brown rings and a yellow halo (Sup. Doc. 1: Sup. Fig. 1). The symptoms agreed with those of Cpa disease of sweetbox reported from Washington D.C. (Salgado-Salazar et al. 2019). Diseased plants were located ~1.5 m from Buxus sempervirens with boxwood blight. Morphological and genetic characterization of isolated fungi and pathogenicity tests followed Salgado-Salazar et al. (2019) (Sup. Doc. 2). White to salmon pink spore masses developed on the abaxial leaf surface after humid chamber incubation. Two distinct fungal cultures were recovered (JAC 18-61, JAC 18-79) on potato dextrose agar (Fisher Scientific, Pittsburg, PA). JAC 18-61 presented cultural and morphological characteristics as described for Cps (Crous et al. 2002). JAC 18-79 produced flat, filamentous, light salmon colonies with tan centers and white filiform borders containing pale pink sporodochia, verticillate and simple conidiophores (x̄: 61.8 ± 20.12 µm, N = 20) with lateral, cylindrical phialides (x̄ = 18.1 ± 5.83 x 2.4 ± 0.7 µm, N = 20), and ellipsoid, hyaline conidia without septa (x̄ = 15.2 ± 1.9 x 3.3 ± 0.7 µm, N = 20). Sexual structures and chlamydospores were not observed. The characteristics of JAC 18-79 agree with those reported for Cpa (Salgado-Salazar et al. 2019). Bidirectional sequencing of the ITS, beta-TUB, and RPB1 and RPB2 regions was performed as described (Salgado-Salazar et al. 2019). BLASTn comparisons against NCBI GenBank revealed JAC 18-61 sequences (MT318150 and MT328399) shared 100% identity with Cps sequences (JX535321 and JX535307 from isolate CB002). Sequences from JAC 18-79 (MT318151, MT341237 to MT341239) were 100% identical to Cpa sequences (MH892596, MH936775, MH936703 from isolate JAC 16-20 and JF832909, isolate CBS 128674). The genome of JAC 18-79 was sequenced and yielded an assembly of 26.3 Mb (204 contigs > 1000 bases, N50 = 264.3 kb, 92x coverage, JABAHV0000000000) that contained the MAT1-2 mating-type idiomorph and shared 98.9% similarity with Cpa BPI910731. Isolate JAC 18-61 (Cps) caused lesions on wounded and unwounded sweetbox and boxwood leaves (Sup. Table 1). In general, JAC 18-79 (Cpa) infected only wounded leaves of both hosts; however, in one trial, one unwounded sweetbox and two unwounded boxwood plants developed lesions, possibly due to the presence of natural wounds. Control plants did not develop symptoms. These results diverge to some degree from previous reports of Cpa infecting unwounded sweetbox and not infecting wounded boxwood (Salgado-Salazar et al. 2019). These results indicate that virulence variation among Cpa isolates might occur. Plating of symptomatic tissue and examination of spores fulfilled Koch's postulates for both pathogens. To our knowledge, this is the first report of Cpa blight on sweetbox in Pennsylvania, and the second U.S. report of the disease. This is also the first report of co-infection of Cpa and Cps on diseased sweetbox foliage. Given the capacity of Cpa to infect both sweetbox and boxwood, inspection for Cpa on both hosts is advisable.

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.

The oospore stage of Plasmopara obducens, impatiens downy mildew.

Impatiens downy mildew is caused by Plasmopara obducens, a pathogen known in the United States for over a hundred years, but newly attacking ornamental Impatiens walleriana in production and in the landscape. Little is known about the life cycle of P. obducens; thus, in this study an attempt was made to determine whether the pathogen is homothallic or heterothallic. Fourteen single-sporangium isolates and three single-zoospore isolates were used in single and dual inoculations of stem tissue to see whether the pathogen was homothallic or heterothallic; all isolates tested were able to produce oospores when inoculated singly, suggesting homothally. Cold treatment at 0 C for at least 1 mo induced oospores to germinate and produce primary sporangia. Inoculation of plant tissue with germinating oospores resulted in infection. Other incubation temperatures (-10, 10, and 20 C) did not induce germination, but fluctuating temperatures (between -10 and 0 C, or 0 and 10 C) induced some germination. Spores incubated at -10 C had significantly thicker walls than spores incubated at other temperatures. Evidence suggests that oospores can serve as an overwintering stage.

Spinach Downy Mildew: Advances in Our Understanding of the Disease Cycle and Prospects for Disease Management.

Downy mildew on spinach is caused by Peronospora effusa, an oomycete pathogen that poses a challenge to spinach production worldwide, especially in organic production. Following infection, P. effusa produces abundant amounts of asexual sporangia. Sporangia become windborne and initiate new infections locally or distantly, leading to widespread epidemics. Oospores produced from the union of opposite mating types have been observed within infected leaves and seeds and may remain viable for many years. Sexual reproduction increases the genetic diversity of P. effusa through sexual recombination, and thus, the movement of oospores on seed has likely fueled the rapid explosion of new pathotypes in different regions of the world over the past 20 years. This review summarizes recent advances in spinach downy mildew research, especially in light of the findings of oospores in contemporary commercial spinach seed lots as well as their germination. Knowledge of the role of the oospores and other aspects of the disease cycle can directly translate into new and effective disease management strategies.

Thermal sensitivity of Calonectria henricotiae and Calonectria pseudonaviculata conidia and microsclerotia.

Knowledge of the thermal sensitivity of conidia and microsclerotia is useful for developing plant disease management approaches that deploy heat to inactivate infectious vegetative propagules of fungal pathogens. For boxwood blight disease, heat treatment of cuttings that harbor conidia and microsclerotia would provide a useful management tool for suppressing the pathogenic activity of Calonectria pseudonaviculata (present in the United States) and C. henricotiae (a quarantine pathogen not present in the United States). In this study, we investigated the thermal sensitivity of conidia and microsclerotia of the boxwood blight pathogens C. henricotiae and C. pseudonaviculata treated in water at 45, 47.5, 50, 52.5, and 55 C. For conidia, as time of exposure increased at each temperature, the proportion of germinated conidia decreased. The predicted time required to inactivate 90% of C. pseudonaviculata conidia (LD90) decreased as water temperature increased from 45 to 55 C and ranged from 35.4 to 5.6 min, respectively. Inactivation of conidia was dependent on isolate, species of Calonectria, and length of exposure at each temperature tested. Microsclerotia of C. henricotiae and C. pseudonaviculata displayed reduced germination with increasing exposure and higher temperatures of hot water. Microsclerotia of C. henricotiae were significantly more resistant to heat treatment than C. pseudonaviculata at 47.5 and 50 C, whereas microsclerotia of both species were rapidly killed at 55 C.

The Effect of Different Temperatures and Moisture Levels on Survival of Calonectria pseudonaviculata in Boxwood Leaves and Twigs and as Microsclerotia Produced in Culture.

Leaves and twig sections of boxwood infected with Calonectria pseudonaviculata were incubated in sand at two moisture levels (36% [carrying capacity] and 5% water [vol/vol]) and at five temperatures (-10, 0, 10, 20, and 30°C). Percent sporulation from monthly tissue samples plated on glucose yeast-extract tyrosine media declined to zero after 5 months at 30°C and after 7 months at -10°C. At 0, 10, and 20°C, sporulation was observed through 30 months. Statistical analysis of data collected over 16 months of sampling showed a significant effect for temperature in all sample types, with maximum survival at 10°C. For discrete microsclerotia grown on cellophane sheets, sporulation was not observed after 2 months at 30°C or after7 months at -10°C. At all other temperatures, they continued to germinate over the 30 months of sampling. Statistical analysis showed significant effects for temperature and moisture level, with maximum survival at 0°C in moist soil over 16 months. These results suggest that extremes of heat and cold will kill the pathogen in plant debris but, at moderate temperatures, it will remain in soil for long periods, making replanting in affected sites in well-moderated climates difficult.

Pyracantha 'Mohave' Fruit Infection by Phytophthora ramorum and Transmission of the Pathogen from Infected Fruit to Roots of Viburnum tinus.

Colonization of the fleshy fruit of Cornus florida, C. kousa, Laurus nobilis, Malus hupehensis, and Pyracantha 'Mohave' was observed following inoculation with sporangia of Phytophthora ramorum. However, abundant production of chlamydospores was only observed in the fruit of Pyracantha 'Mohave'. Pyracantha 'Mohave' fruit that had been inoculated with a P. ramorum sporangia suspension were placed in pots containing rooted cuttings of Viburnum tinus in a misting tent or in water-filled trays in a climate-controlled greenhouse. Runoff was collected for 24 to 30 days, and roots were plated after the final collection. Mean percent recovery from infected roots was not significantly different (P = 0.05, Tukey's test) between bottom-watered treatments in trays and misted treatments, averaging 58% for bottom-watered and 54% for mist treatments. The number of CFU collected in runoff from bottom-watered plants was consistently lower than that obtained from plants held under mist, likely due to desiccation of the fruit. The results show that root infection of V. tinus can occur by P. ramorum via infected fruit of Pyracantha 'Mohave'. This phenomenon represents a pathway of infection for P. ramorum not previously reported, which may play a role in disease epidemiology.

Susceptibility of Some Common Container Weeds to Phytophthora ramorum.

Phytophthora ramorum is known to infect a number of ornamental plants grown in containerized culture. However, pots may also contain weeds. In this research, the foliage of 14 common weeds of containerized plant culture was inoculated with P. ramorum to determine susceptibility of aboveground parts. Three species were found to develop leaf lesions: northern willowherb (Epilobium ciliatum), fireweed (Chamerion angustifolium), and a fern (Pteris cretica). Weed roots from 11 species were inoculated to see if P. ramorum could persist on roots, and P. ramorum was isolated from most plant roots 1 month after inoculation when the washed roots were plated on selective medium; they were recovered only to a minor extent from surface-sterilized roots of weeds. Additional experiments were done to collect and sample runoff from pots containing inoculated plants to see if inoculum was produced on weed roots. In these experiments, little inoculum was found in runoff from root-inoculated weeds compared to Viburnum tinus. Percent root colonization recorded from washed roots was significantly greater in Viburnum compared to the weeds, and weeds that were foliar hosts had greater root colonization than weeds that were not.

A test system to quantify inoculum in runoff from Phytophthora ramorum-infected plant roots.

Foliar hosts of Phytophthora ramorum are often susceptible to root infection but the epidemiological significance of such infections is unknown. A standardized test system was developed to quantify inoculum in runoff from root-infected Viburnum tinus ?Spring Bouquet? or Rhododendron ?Cunningham's White? cuttings. Cuttings of both species gave off a maximum amount of inoculum 1 to 3 weeks after inoculation. The greatest amount of inoculum was recovered from Viburnum roots that were 48 to 70 days old at the time of inoculation, or roots incubated at 15 to 20?C rather than 25?C. Inoculum in runoff from inoculated Viburnum roots was similar for four different isolates of P. ramorum representing both the NA1 and EU1 lineages. When Rhododendron cuttings were inoculated with P. ramorum, P. citricola, or P. cactorum, inoculum of all three pathogens was recovered from runoff, with the highest amount recovered from plants inoculated with P. citricola, followed by the other two. Compared with the other two pathogens, P. ramorum colonized root tissue to a smaller extent. The epidemiology of root infection by P. ramorum is important in itself but the assay might lend itself for use in risk analysis for root infection of other plant species and evaluation of control measures, and also shed light on other root-infecting Phytophthora spp.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Propagule Production by Phytophthora ramorum on Lilac (Syringa vulgaris) Leaf Tissue Left on the Surface of Potting Mix in Nursery Pots.

Lilac leaf tissue infected with Phytophthora ramorum was placed on top of potting mix in pots and exposed to different watering regimes or different temperatures to determine if it could serve as a source of inoculum. If pieces of infected leaf were placed in pots containing healthy lilac plants kept under constantly moist conditions or under twice-a-day trickle irrigation for 1 month, inoculum production from infected tissue declined for the first 4 days but declined significantly less steeply under constantly moist conditions. At the end of the experiment, 28% of plants exposed to moist conditions developed root infections, whereas only 6% exposed to trickle irrigation did. If infected leaf pieces were placed on the surface of potting mix in pots containing lilacs and watered for 5 min one, two, or three times a day, inoculum production in the first 4 days declined but declined significantly more slowly in pots watered three times a day. If 0 to 16 leaf pieces were placed on the surface of potting mix in pots containing healthy lilacs under constantly moist conditions, leaf number significantly influenced the incidence of root infection. The effect of temperature was more difficult to quantify. At 10 or 15°C, propagules included zoospores whereas, at 20 or 25°C, they were predominantly sporangia. These results confirm the importance of detached leaves as inoculum producers under greenhouse conditions.

Persistence of Phytophthora ramorum in Soil Mix and Roots of Nursery Ornamentals.

Although most Phytophthora species have a soilborne phase that is crucial for infection of roots and for survival away from the host, the details of the soil phase of Phytophthora ramorum are not yet fully understood. As mycelium ages, it becomes resistant to sterilization by acidic electrolyzed water (AEW), a product of the electrolysis which can be used as a disinfectant. Colonies of P. ramorum could be recovered from moist potting mix or sand for many months, whether buried as infected plant leaf tissue or as mycelium bearing chlamydospores, and the buried material was also resistant to treatment by AEW. There was no significant difference in recovery over time among treatments (sand or potting mix; infected plant tissue or mycelium); after approximately a year, colonies could be recovered at 0.8 to 14.3%. When excised roots were inoculated with P. ramorum sporangia and buried in mesh bags in potting mix, the pathogen was recovered from buried roots for at least 8 to 11 months, but it was not clear whether it was surviving as mycelium or chlamydospores. The roots of living plants of Acer macrophyllum, Buxus sempervirens, Camellia oleifera, C. sinensis, C. sasanqua, Lonicera hispidula, Taxus baccata, Umbellularia californica, Vaccinium macrocarpon, Viburnum davidii, V. tinus, V. × pragense, Rhododendron 'Gloria', and Syringa vulgaris were drenched with a sporangial solution of P. ramorum and incubated for a month; the pathogen could be recovered from roots of all plants except those of Buxus sempervirens and Lonicera hispidula. Recovery on selective agar medium (P5ARP) was from both washed and surface-sterilized roots, suggesting that the roots were internally infected. When chlamydospores were placed near roots and observed directly, they were seen to germinate, forming sporangia. Nearby roots became infected, the tips covered with sporangia. Therefore, P. ramorum appears to have a soil phase, at least under greenhouse and nursery conditions.

Resistance to Triadimefon and Benomyl: Dynamics and Impact on Managing Cucurbit Powdery Mildew.

Frequency of fungicide-resistant strains of Podosphaera xanthii on pumpkins in New York before treatment varied from 3 to 80% for the demethylation inhibiting (DMI) fungicide triadimefon and from 0 to 48% for the benzimidazole fungicide benomyl between 1993 and 1996. When the initial frequency of triadimefon-resistant strains was less than 55%, one application of triadimefon plus chlorothalonil was effective. This application was made after reaching the action threshold of one leaf with powdery mildew symptoms per 50 old leaves (defined as the oldest third of the foliage). The frequency of triadimefon-resistant strains increased from 3 to 71% by 20 days after the first fungicide application in 1993. Triadimefon in the second application did not contribute to control. Loss of efficacy was due to resistance because, compared with triadimefon-treated pumpkins, pumpkins treated with other systemic fungicides were less severely infected by powdery mildew on abaxial leaf surfaces where the companion multi-site contact fungicide contributes little to control. Triadimefon was not effective in 1995 when 80% of the pathogen population was resistant before treatment. Benomyl was effective in 1995, but not in 1996 when 48% of the isolates tested were resistant to both benomyl and triadimefon before treatment. An in-field seedling assay was developed to determine local occurrence of resistant strains before the first treatment was needed. Although sensitivity of the pathogen population to the DMI fungicides myclobutanil and propiconazole also decreased after they were applied, these fungicides were more effective than triadimefon.