Boxwood Cultivars Research Articles

Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants

BackgroundAnti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars ‘Vardar Valley’ and ‘Justin Brouwers’ on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification.ResultsBacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted.ConclusionThis study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity.

Combating an Invasive Boxwood Pathogen - Calonectria pseudonaviculata - in the United States by Shifting Production to Less Susceptible Cultivars.

Boxwood Blight (BB) caused by Calonectria pseudonaviculata (Cps) is an economically devastating disease affecting the entire boxwood supply chain from growers to gardeners, since it was first officially documented in the United States in 2011. This disease has taken a heavy toll on boxwood, an iconic landscape plant and the number one evergreen nursery crop. The objective of this study was to examine the adoption of one sustainable management strategy available to growers: shifting boxwood production from highly susceptible to less susceptible cultivars. We investigated the ongoing shift by comparing boxwood sales of 17 selected nurseries from seven states across the country in 2011, 2016 and 2021. Results revealed that from 2021 to 2016, sales of cultivars highly susceptible to BB were reduced by over 35% while less sales of less susceptible boxwood cultivars increased 55%. Increased boxwood sales have been seen for 'Winter Gem', 'Wintergreen', 'SB 300' (Freedom®), 'SB 108' (Independence®), and 'Little Missy', all of which have been rated less susceptible than B. sempervirens 'Suffruticosa' in numerous trials. The potential for long-term positive impact on sustainable boxwood production and plantings in the U.S. through the use of such less susceptible cultivars is discussed. Better boxwood cultivar choices will build crop health into new plantings and sustain customer demand for boxwood. This is a case study for how sustainable crop protection strategy helps to maintain production of a crop under serious pressure from an invasive pathogen.

Multi-Year Field Plantings Evaluating Boxwood Cultivars for Susceptibility to the Blight Pathogens (Calonectria spp.) in Northern Germany.

Two multiyear field trials were conducted to evaluate boxwood cultivars for their susceptibility to the blight pathogens Calonectria pseudonaviculata and C. henricotiae in northern Germany. Fifteen cultivars were included in the first trial from 2007 to 2012, and 46 cultivars were included in the second trial from 2014 to 2017. Both trials were done in a naturally infested field that was supplemented with infected plant tissue added to the soil before planting. Each cultivar had three replicate hedge sections with 10 plants per section, and they were assessed annually for blight severity expressed as proportion of leaves blighted and fallen. Blight severity varied significantly among years (P < 0.0001) and cultivars (P < 0.05) within each trial. In the first trial, mean severity ranged from 0.03 to 0.11 for the most resistant cultivars and 0.35 to 0.96 for the most susceptible ones. Similarly, in the second trial, mean severity ranged from 0.06 to 0.27 and 0.71 to 0.97 for the most resistant and susceptible cultivars, respectively. 'Suffruticosa' was consistently the most susceptible cultivar, followed by 'Marianne', 'Myosotidifolia', 'Raket', and 'Morris Midget'. 'Herrenhausen' was the most resistant cultivar, followed by B. microphylla var. japonica, B. microphylla var. koreana, 'Green Mound', 'Faulkner', and 'Winter Beauty'. This study provides field data showing the performance of boxwood cultivars under different levels of disease pressure in an area where C. henricotiae was dominant. This knowledge will help boxwood growers and gardeners to choose less susceptible cultivars and help plant breeders to select for disease resistance.

The effect of temperature, leaf wetness period, and cultivar susceptibility on boxwood blight disease development and sporulation.

Boxwood blight causes great losses to the boxwood nursery industry and landscapes in 30 states in the USA. Understanding the epidemiological factors governing disease development will be important for disease forecasting and design of best management practices. We evaluated the effect of leaf wetness period (lwp) and temperature on lesion development and sporulation on three boxwood cultivars under controlled conditions to develop predictive models for disease development. We conducted detached leaf assays at 18 to 27°C, and various lwp, with the cultivars Buxus sempervirens 'Suffruticosa' (highly susceptible), Buxus × 'Green Velvet' (moderately susceptible), and B. microphylla var. japonica 'Winter Gem' (less susceptible). Detached leaves were inoculated with 200 conidia in 50 µL of suspension and disease incidence was recorded at 3 - 13 d post inoculation (dpi). Cultivar, lwp, temperature, and most interactions significantly influenced disease development. A minimum of 5 h of leaf wetness was required for any disease. Lesion development increased most rapidly between 12 - 15 h and continued to increase to about 21 h of leaf wetness. Temperatures between 21 and 25°C were optimal for lesion development. There was about a 7-d lag between appearance of lesions and maximal incidence of sporulation. The two less-susceptible cultivars had fewer lesions than 'Suffruticosa' under the same infection conditions; in addition, leaf lesions of 'Winter Gem' exhibited delayed sporulation and sporulation from a smaller proportion of symptomatic leaves. Response surfaces were developed for each cultivar to predict the disease incidence using the lwp and dpi. Our findings will help refine disease forecast models to improve management of boxwood blight.

Building Health into New Boxwood Crops and Plantings by Making Informed Cultivar Selection

Boxwood blight caused by Calonectria pseudonaviculata and leafminer ( Monarthropalpus flavus) are the primary boxwood disease and pest, respectively. This review summarizes the latest studies evaluating boxwood cultivars and selections against boxwood blight and leafminer in the hopes of facilitating adoption of identified cultivars with resistance to both, fast tracking the nation onto a more sustainable path toward healthy production and gardening of this iconic landscape plant.

Screening of Existing and New Boxwood Cultivars for Disease Resistance and In Vitro Fungicide Screening to Manage Boxwood Dieback Caused by Colletotrichum theobromicola

Boxwood (Buxus spp. L.) is one of the most common and widely planted perennial ornamentals in both home gardens and commercial landscapes. Although considered robust, boxwood is susceptible to several plant pathogens. Boxwood dieback, a recently reported fungal disease caused by Colletotrichum theobromicola, has been spreading rapidly within the United States. Symptoms of boxwood dieback are difficult to distinguish visually in affected plants due to the symptom resemblance to other known biotic and abiotic diseases of boxwood. Previous studies have shown that it may take 2 to 3 months for the symptoms to appear under greenhouse conditions. Although a molecular method has been designed for early, rapid, and accurate pathogen detection, boxwood breeders, nursery growers, and landscape professionals are concerned about the lack of effective management practices. Therefore, the primary objectives of this study were to devise effective disease management strategies by screening cultivars to determine their susceptibility to boxwood dieback and screening various fungicides to determine their effectiveness in managing the disease. Host range studies were conducted by screening a wide variety of boxwood cultivars under greenhouse conditions. The boxwood cultivar ‘Little Missy’ showed much-delayed symptom expression compared with the rest of the cultivars. None of the 11 cultivars were found to be resistant to boxwood dieback. In vitro screening of nine fungicide treatments was conducted to determine inhibition of mycelial growth and spore germination for eight isolates of C. theobromicola collected from eight states in the United States. Of the nine fungicide treatments, difenoconazole + pydiflumetofen showed maximum inhibition of mycelial growth and spore germination at 1 ppm active ingredient, followed by fluxapyroxad + pyraclostrobin, and pyraclostrobin + boscalid at 5 ppm active ingredient. Azoxystrobin + benzovindiflupyr significantly inhibited mycelial growth at 1 ppm but reduced spore germination only at 10 ppm active ingredient. This study provides boxwood industry professionals with critical information pertaining to host susceptibility and fungicide efficacy that may be applied to effectively mitigate boxwood dieback and reduce its further spread.

Significance to the Horticulture Industry

Significance to the Horticulture Industry

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.

Endophytic Burkholderia sp. SSG as a potential biofertilizer promoting boxwood growth.

BackgroundBurkholderia sp. SSG is a bacterial endophyte isolated from boxwood leaves showing a resistant response to infection by the boxwood blight pathogen Calonectria pseudonaviculata. SSG acted as a protective and curative biocontrol agent for boxwood blight and as a bio-sanitizer of disease inoculum in the field. Many gene clusters involved in antibiotic production and plant growth promotion (PGP) were found in the genome, giving this endophyte great application potential as a treatment for plant protection. However, the PGP features have not been documented. This study investigated the plant growth promotion activity of SSG in boxwood.MethodsTo determine whether SSG is a plant growth promoting bacterium, four PGP traits, auxin and siderophore production, nitrogen fixation and phosphate solubilization, were examined in the laboratory with colorimetric or agar plate assays. The plant growth promoting activity of SSG was tested on three boxwood varieties characterized by slow, intermediate and fast growth rates, namely Justin Brouwers, Buddy and Winter Gem, respectively. These plants were drenched with an SSG cell suspension or water and washed plant weight was compared before and after treatment to determine growth changes after 10 months.ResultsThe SSG culture was sustainable on nitrogen free media, suggesting that SSG may fix atmospheric nitrogen. It was also a strong phosphate solubilizer and a potent siderophore and indole-3-acetic acid (IAA) producer. Significant growth promotion was observed on boxwood cultivars Justin Brouwers, Buddy and Winter Gem 10 months after plant roots were drenched with SSG cells. The growth rate of treated plants was 76.1, 58.3, and 37.3% higher than that of the control, respectively. The degree of growth promotion was significantly different among plant varieties, notably more pronounced with the slow and intermediate growers. This study demonstrates that the SSG bacterium has multiple PGP traits and is a prospective plant biofertilizer.

Curative Fungicide Activity Against Calonectria pseudonaviculata, the Boxwood Blight Pathogen1

Abstract Azoxystrobin, azoxystrobin plus benzovindiflupyr, kresoxim-methyl, propiconazole, pyraclostrobin, pyraclostrobin plus fluxapyroxad, tebuconazole, tetraconazole, thiophanate-methyl, and triflumizole fungicides were evaluated for curative and anti-sporulant activity against boxwood blight caused by Calonectria pseudonaviculata on detached leaves and whole boxwood plants (Buxus spp.). Pretreating detached leaves with 30 or 300 ppm a.i. 24 h prior to inoculation reduced disease compared to the untreated control for all fungicides. Fungicides were also applied 24 to 96 h post-inoculation. Only propiconazole reduced diseased leaf incidence to at least half of the control. When leaves were treated post-infection with 300 ppm propiconazole, tetraconazole, tebuconazole, or triflumizole, the pathogen did not sporulate over 2 wks. Propiconazole also reduced the percent of leaf area diseased; lesions were nearly 80% smaller with 300 ppm applied 48 h after inoculation. ‘True Dwarf' boxwood plants treated with 450 ppm thiophanate-methyl, 120 ppm pyraclostrobin or 150 ppm propiconazole 48 h after inoculation demonstrated that only propiconazole reduced the number of diseased leaves, blight lesions and the frequency of pathogen re-isolation. Experiments with ‘Green Mound' and ‘Green Mountain' boxwood cultivars and additional fungicides applied 48 h after inoculation demonstrated that propiconazole at 300 ppm, pyraclostrobin plus fluxapyroxad (150 ppm each) and azoxystrobin (135 ppm) plus benzovindiflupyr (67.5 ppm) reduced disease. Index words: fungicide management, Buxus, chemical disease management Chemicals used in this study: azoxystrobin (Heritage 50 WG), azoxystrobin plus benzovindiflupyr (Mural 30, 50 WG), kresoxim-methyl (Cygnus 50 WG), propiconazole (ProCon-Z 14.3 L), pyraclostrobin (Insignia 20 WG), pyraclostrobin plus fluxapyroxad (Orkestra Intrinsic 21.26 SC), tebuconazole (Torque 38.7 SC), tetraconazole (Minerva 11.6 SC), thiophanate-methyl (3336 50% WP), triflumizole (Procure 480 SC). Species used in this study: boxwood (Buxus L.), boxwood blight (Calonectria pseudonaviculata (Crous, J.Z. Groenew. &amp; C.F. Hill) L. Lombard, M. J. Wingf. &amp; Crous.

Ranking Resistance of Buxus Cultivars to Boxwood Blight – an Integrated Analysis1

Abstract Boxwood (Buxus L. spp., Buxaceae) are popular woody landscape shrubs grown for their diverse forms and broad-leaved evergreen foliage, with an estimated $126 million economic impact in the U.S. alone. Boxwood plants grown in temperate zones worldwide are now threatened by a destructive blight disease caused by the ascomycete fungi, Calonectria pseudonaviculata and C. henricotiae. While the disease can be mitigated somewhat through cultural practices and fungicides, the most sustainable long-term solution is the development of disease-resistant boxwood cultivars. Hundreds of boxwood accessions from the National Boxwood Collection at the U.S. National Arboretum were screened for resistance using a lab-based, detached-leaf assay. Initial comparisons of our results with those of multiple other disease resistance assays found inconsistent ranking of cultivar resistance among studies. We used a meta-analysis approach on compiled data from six studies and were able to produce a consistent ordering of cultivars sorted by their susceptibility to boxwood blight, despite the diversity in materials and methods of the studies. Index words: Boxwood, Calonectria pseudonaviculata, Cylindrocladium buxicola, meta-analysis, plant breeding, resistance screening. Species used in this study: Buxus bodinieri H. Lev.; B. harlandii Hance; B. microphylla Seibold &amp; Zucc.; B. sempervirens L.; B. sinica var. insularis (Nakai) M. Cheng; B. wallichiana Baill.; Calonectria pseudonaviculata (Crous, J.Z. Groenew. &amp; C.F. Hill) L. Lombard M.J. Wingf. &amp; Crous 2010.

Susceptibility of Boxwood Accessions from the National Boxwood Collection to Boxwood Blight and Potential for Differences between Calonectria pseudonaviculata and C. henricotiae

Forty Buxus accessions from the U.S. National Arboretum National Boxwood Collection were evaluated as potted plants and detached leaves for susceptibility to Calonectria pseudonaviculata (Crous et al.) L. Lombard et al., and nine boxwood cultivars were evaluated against both species of Calonectria causing boxwood blight, C. pseudonaviculata and C. henricotiae. Accessions of B. harlandii Hance, B. sinica (Rehder and E.H.Wilson) M.Cheng, and B. microphylla Siebold and Zucc. had less disease than B. microphylla ×sempervirens, and all had fewer lesions per plant than the 20 B. sempervirens L. accessions evaluated. Variation within species was observed. Of the individual accessions, B. sinica var. aemulans (accession 60705*H), B. sempervirens (36365*J), and B. harlandii (18834*H) were least susceptible, with &lt;10 lesions per plant. B. sempervirens ‘Scupi’ (9548*H), B. microphylla ‘Compacta’ (4899*CH), B. sempervirens ‘Arborescens’ (57953*H), B. sinica var. insularis ‘Pincushion’ (51898*H), and B. microphylla var. japonica ‘Jim Stauffer’ (72213*H) each had &lt;20 lesions. These rankings differ from previous studies that used detached leaf and unrooted cutting assays. Normalizing to account for plant size effects on inoculation and disease increased variability for individual accession rankings but did not result in significant differences in the most and least susceptible accessions or species ranking. Nine boxwood cultivars evaluated against both pathogen species exhibited a range of susceptibility against four pooled isolates each of C. pseudonaviculata and C. henricotiae. Although small differences in disease severity were observed on boxwood inoculated with the two pathogens, there was no interaction of cultivar and pathogen species, suggesting that a cultivar rated resistant to one species was resistant to the other. These results may aid boxwood breeders to develop resistance to boxwood blight.

Effects of Inoculum Dose, Temperature, Cultivar, and Interrupted Leaf Wetness Period on Infection of Boxwood by Calonectria pseudonaviculata.

Boxwood blight is an emerging disease of great concern for the ornamental horticulture industry, historic garden managers, landscapers, and homeowners. Controlled-environment experiments were conducted to determine the effects of conidial concentration, temperature, interrupted leaf wetness period, cultivar, and leaf age on infection of boxwood leaves by Calonectria pseudonaviculata. Boxwood blight incidence (BBI) increased with increasing concentration up to 2.0 × 104 spores/ml. BBI also increased as temperature increased from 18 to 25°C, then declined gradually to zero at 29°C. Similar infection effects of inoculum concentration were observed in an experiment with four boxwood cultivars ('Justin Brouwers', 'John Baldwin', 'Green Mound', and 'Nana') of various degrees of susceptibility. The hypothesis that younger leaves are more susceptible than older leaves was supported for Justin Brouwers and Nana but not for Green Mound; and younger leaves of John Baldwin were less susceptible than older leaves. When inoculated plants ('Suffruticosa') were exposed to dry interruptions of 3 h or longer between 5 or 8 h of initial wetness and 12 h of additional wetness, these plants had significantly lower BBI compared with those exposed to continuous wetness for 20 h, and similar or at most slightly more infection than plants exposed to only the 5- or 8-h initial wetness. Continuous wetness durations beyond 20 h did not increase infection in these experiments. These results advanced our understanding of the environmental requirements of the infection process in boxwood blight development and they are essential for refining disease forecasting models.

Use of Mycelium and Detached Leaves in Bioassays for Assessing Resistance to Boxwood Blight.

Boxwood blight caused by Calonectria pseudonaviculata is a newly emergent disease of boxwood (Buxus spp. L.) in the United States that causes leaf drop, stem lesions, and plant death. A rapid and reliable laboratory assay that enables screening hundreds of boxwood genotypes for resistance to boxwood blight is needed to enable breeding and selection of resistant cultivars. Using eight boxwood cultivars with differing susceptibilities, we examined parameters for a screening assay comparing whole plant inoculation with detached leaf inoculation, use of mycelium versus spores as the inoculum, comparison of times of the year for inoculation, and comparison of two leaf inoculation methods. Inoculation of detached leaves gave comparable results to inoculation of whole plants when compared across genotypes, although the detached leaf assay resulted in greater percentages of symptom expression. The time of year of plant inoculation (spring, summer, or winter) did not affect the relative expression of symptoms among the most resistant and susceptible genotypes. Inoculating plants with mycelium was as effective as spore inoculation for causing disease symptoms and allowed us to distinguish the more resistant genotypes, yet mycelium inoculation was much easier to prepare in large quantities for multiple assays.

Management of Calonectria pseudonaviculata in Boxwood with Fungicides and Less Susceptible Host Species and Varieties.

Calonectria pseudonaviculata causes leaf and stem lesions resulting in defoliation and dieback of boxwood. Trials were conducted to evaluate fungicide management of boxwood blight under greenhouse and container nursery conditions in Connecticut using fungicides previously determined to have in vitro activity against conidial germination or mycelial growth. Plants of different boxwood cultivars were inoculated 48 h after fungicide application. Disease progression was monitored over 6 weeks and progressed from leaf and stem lesions to defoliation. The level of disease control achieved by fungicides was generally good, with the most efficacious treatments averaging from 95 to nearly 100% control. Products containing propiconazole, myclobutanil, thiophanate-methyl, fludioxonil, pyraclostrobin, kresoxim-methyl, and chlorothalonil had significant efficacy. The combination of systemic plus protectant fungicides in a single application resulted in superior disease control compared to the use of a systemic fungicide. There were no differences between the fungicide treatments that included thiophanate-methyl and those that included propiconazole as the systemic fungicide. Korean and 'Winter Gem' (Buxus sinica var. insularis) were the least susceptible of the cultivars evaluated, common boxwood (B. sempervirens) and True Dwarf (B. sempervirens 'Suffruticosa') were the most susceptible, and 'Green Mountain' (B. sinica var. insularis × B. sempervirens Suffruticosa) and 'Green Velvet' (B. sinica var. insularis × B. sempervirens Suffruticosa) were intermediate. These results suggest that B. sinica var. insularis may have some level of resistance to boxwood blight. Management of boxwood blight will rely on integrated best management practices that include inspection of incoming plant material, sanitation, cultural controls including use of cultivars tolerant to infection, and fungicide application.

Boxwood Blight: A New Scourge, a New Paradigm for Collaborative Research

boxwood prominence The first boxwoods were introduced to the United States in the 1650s and have been an important element in landscape design ever since, from colonial gardens to Beatrix Farrand’s 1921 design for the grounds of Dumbarton Oaks, to the knot gardens of today. Boxwoods form buds on old wood in addition to new stems, making them easy to shape as hedges or topiary, and their slow growth and the fine texture of their foliage make them ideal as foundation plantings and as structural backdrops in formal gardens. As slow-growing plants, they are both difficult and profitable for the nursery industry. The current wholesale market value for boxwood nursery production is estimated to be $103 million annually (United States Department of Agriculture, 2010). Major boxwood production states include Oregon, with approx. 200 nurseries growing a variety of boxwood cultivars (Oregon Department of Agriculture, 2012), selling $22 million in 2009, according to the 2010 USDA-NASS Census of Horticulture; California, Texas, Ohio, and Illinois sold $9.5, $8.2, $7.3, and $6.7 million of boxwood, respectively (United States Department of Agriculture, 2010).

Pseudonectria buxi causing leaf and stem blight on Buxus in Canada

An outbreak of a boxwood disease was observed in nurseries in southern Ontario in 2008, but the disease appears to have been present in Ontario for at least 15 years. From 2008 to 2010, over 300 fungal isolates were obtained from diseased samples, which included cuttings and whole plants. Almost half of the isolates (144) were found to belong to a single cultural morphotype which was confirmed as Pseudonectria buxi by DNA sequencing of several isolates. The remaining isolates were grouped into another seven major morphotypes, the largest of which accounted for 11 % of the isolates. Single representatives of all eight morphotypes were identified to species using morphological and molecular techniques, and were subjected to pathogenicity testing. Among them, only Pseudonectria buxi successfully satisfied Koch’s postulates on detached leaves, small twigs and rooted cuttings, which confirmed it as the causal agent of Volutella blight. Inoculation tests showed that wounds may be the major infection courts for P. buxi, since non-wounded inoculated tissues did not become diseased. Among several commonly grown boxwood cultivars in Ontario, ‘Green Gem’ was the most susceptible compared to ‘Green Velvet’, ‘Green Mound’, ‘Green Mountain’ or ‘Pincushion’, but all were found to be susceptible. Rather than behaving just as a saprophyte or secondary invader, P. buxi is capable of primary invasion after wounding, and causing extensive disease on live boxwood tissues.

Growth, Survival, and Aesthetic Quality of Boxwood Cultivars as Affected by Landscape Exposure

Three cultivars of boxwood, `Winter Gem' (Buxus microphylla Sieb. &amp; Zucc.), `Green Velvet', and `Green Mountain' [B. sinica (Rehd. &amp; Wils.) Cheng var. insularis (Nakai) M. Cheng × B. sempervirens L. `Suffruticosa' L.], were planted in 12 different landscape exposures (N, ENE, NNE, E, SSE, ESE, S, WSW, SSW, W, NNW, WNW) at Manhattan and Wichita, Kans., representing U.S. Dept. of Agriculture hardiness zones 5 and 6, respectively. In Kansas, winter stress is often the greatest threat to plant survival, and Winter 1995-96 was one of great extremes. Official lows of -25 °C for Manhattan and -23 °C for Wichita were recorded, along with sharp 24-hour temperature drops of 31-32 °C in January and March. Differences in cultivar performance were noted between sites. Better winter quality was exhibited by `Green Velvet' and `Green Mountain', although significant bronzing occurred on the former in Manhattan. Locations on N, ENE, NNE, NNW, and WNW exposures resulted in better plant quality of all cultivars, whereas plant performance was poorer at S and SE exposures. Differences in canopy temperatures of up to 15.7 °C were recorded, on the same cold winter day, between NNW and ESE. Recovery from winter dieback and growth during Summer 1996 varied with cultivar, site, and exposure. `Winter Gem' grew most in height, and `Green Velvet' grew most overall at Manhattan. Growth rates were dependent on exposure, with greater growth at NNE, E, SSW, and WNW. Performance of `Green Velvet' and `Green Mountain' indicated that they could be used in any exposure with conditions similar to those of the test sites.

Hardiness and Aesthetic Quality of Boxwood Cultivars as Affected by Landscape Exposure

Five cultivars of boxwood (Buxus microphylla)—'Winter Gem', B. microphylla var. japonica `Green Beauty', `Green Velvet', `Green Mountain' and `Glencoe'—were planted in twelve different exposures at Manhattan and Wichita, Kan., representing USDA hardiness zones 5 and 6 respectively. The 1995–96 winter was one of great extremes. Lows of –25°C for Manhattan and -23°C for Wichita were recorded, along with sharp 24-hour temperature drops of 31–32°C in January and March. Differences in cultivar performance were noted between the sites. At the Wichita site best winter color was exhibited by `Green Velvet' and `Glencoe', whereas `Green Mountain' sustained some bronzing of foliage due to winter sun. At Manhattan only `Glencoe' in protected locations exhibited good winter color. All other surviving cultivars showed considerable bronzing. In addition, `Green Beauty' was severely damaged at Manhattan, sustaining bark splitting due to low temperatures, although most plants survived at Wichita. Shaded locations on north, northeast and northwest produced best plant quality of all cultivars; whereas, the poorest plant performance occurred on south and southeast exposures.

Validation of Boxwood Cultivars by Biochemical Markers

Leaf tissue from 10 Buxus spp. cultivars were collected and evaluated for differences in isozyme and random amplified polymorphic DNA (RAPD) markers. In addition, samples of named cultivars were collected from multiple locations to estimate the degree of cultivar fidelity across locations. In general, isozymes were highly polymorphic among cultivars. Aconitase, malate dehydrogenase, aminoaspartate transferase, and phosphoglucose isomerase were the most useful isozymes to distinguish among the selected clones. Good quality DNA was readily extracted from leaf tissue using a standard CTAB procedure. RAPDs were generated from polymerase chain reactions using a random set of primers. Of 200 primers that were screened, 20 gave reproducible and polymorphic patterns that could be used to distinguish among the boxwood cultivars. In general, cultivar fidelity among commercial nurseries was fair. Biochemical analyses could be used to resolve problems with cultivar identity once baseline information is collected.