Structurally diverse triterpenoid alkaloids from Buxus rugulosa.

Four new nortriterpenoid alkaloids, namely buxrugulines E-H (1-4), along with five known ones (5-9), were isolated from the twigs and leaves of Buxus rugulosa. Their structures were identified based on extensive NMR data and MS spectroscopic analyses. Our bioassays revealed that compounds 5, 6 and 8 exhibited potent cytotoxicity in vitro against MCF-7 cell lines, with IC50 values ranging from 6.70 to 11.00 µM, respectively.

Analgesic Buxus alkaloids with Enhanced Selectivity for the Low-Voltage-Gated Calcium Channel Cav 3.2 over Cav 3.1 through a New Binding Mode.

Low-voltage-gated calcium channels (LVGCCs; Cav 3.1-3.3) represent promising drug targets for epilepsy, pain, and essential tremor. At present, modulators with heightened selectivity for a subtype of LVGCCs are still highly desired. In this study we explored three classes of Buxus alkaloids and identified 9(10/19)abeo-artanes Buxusemine H and Buxusemine L (BXSL) as an unprecedented type of Cav 3.2 inhibitors. Particularly, BXSL exhibited Cav 3.2 inhibition comparable to Z944, a non-subtype-selective LVGCCs inhibitor under clinical trial. While lacking specificity for Cav 3.3, BXSL showed a 30-fold selectivity of Cav 3.2 over Cav 3.1. As compared to several well-known inhibitors, the experimental and computational studies suggested BXSL exhibits a distinct binding mode to Cav 3.2, notably through the essential interaction with serine-1543 in domain III. Furthermore, BXSL showed minimal impact on various recombinant and native nociceptive ion channels, while significantly reducing the excitability of isolated mouse dorsal root ganglion neurons. Animal studies in wild-type and Cav 3.2 knock-out mice revealed that BXSL (5 mg/kg), by inhibiting Cav 3.2, exhibits an analgesic effect equivalent to Z944 (10 mg/kg) or mibefradil (10 mg/kg). Moreover, we proposed a structural rationale for the high selectivity of 9(10/19)abeo-artane-type alkaloids towards Cav 3.2 over Cav 3.1. This study introduces a novel analgesic agent and valuable molecular insight for structure-based innovative Cav 3.2 drug development.

Hazardous waste management (Buxus papillosa) investment for the prosperity of environment and circular economy: Response surface methodology-based simulation.

Dealing with the current defaults of environmental toxicity, heating, waste management, and economic crises, exploration of novel non-edible, toxic, and waste feedstock for renewable biodiesel synthesis is the need of the hour. The present study is concerned with Buxus papillosa with seeds oil concentration (45% w/w), a promising biodiesel feedstock encountering environmental defaults and waste management; in addition, this research performed simulation based-response surface methodology (RSM) for Buxus papillosa bio-diesel. Synthesis and application of novel Phyto-nanocatalyst bimetallic oxide with Buxus papillosa fruit capsule aqueous extract was advantageous during transesterification. Characterization of sodium/potassium oxide Phyto-nanocatalyst confirmed 23.5 nm nano-size and enhanced catalytic activity. Other characterizing tools are FTIR, DRS, XRD, Zeta potential, SEM, and EDX. Methyl ester formation was authenticated by FTIR, GC-MS, and NMR. A maximum 97% yield was obtained at optimized conditions i.e., methanol ratio to oil (8:1), catalyst amount (0.37 wt%), reaction duration (180 min), and temperature of 80 °C. The reusability of novel sodium/potassium oxide was checked for six reactions. Buxus papillosa fuel properties were within the international restrictions of fuel. The sulphur content of 0.00090% signified the environmental remedial nature of Buxus papillosa methyl esters and it is a highly recommendable species for biodiesel production at large scale due to a t huge number of seeds production and vast distribution.

Investigating DNA barcodes of plants growing in some areas of Iran with high crime rate: Quercus brantii, Curpressus arizonica, Crataegus pentagyna, Ziziphus Spina-chtista, and Buxus hyrcana.

According to criminal botany, the offender unknowingly carries plant samples from the crime scene. Therefore, studying the genetic data of plants native to the crime scene can solve many ambiguities in the criminal files. In this regard, the aim of this study was to investigate the genome of 5 endemic plants in some areas of Iran with high crime rate. Quercus brantii, Curpressus arizonica, Crataegus pentagyna, Ziziphus Spina-chtista, and Buxus hyrcana were assessed using 1 genetic fragment on plastid regions (trnH-psbA) as well as 1 gene on nuclear chromosome called ITS. The alignment of DNA sequences of trnH-psbA and ITS genes was done using BioEdit, Clustal X, and Muscle v4.0 software programs. The phylogenetic analysis was performed on aligned data using Maximum Parsimony (MP) and the Bayesian methods. The Splits Tree v.4.14.4 software program was used for phylogenetic network analysis. Finally, the data combinability test was conducted using the Incongruence Length Difference (ILD) test by PAUP* software program. All data from nrDNA ITS and trnH-psbA sequences were consistent with Information Compatibility Test (ICT) results. Moreover, the nrDNA ITS indicated more resolved relationship than trnH-psbA. The results from MP and Bayesian analyses did not differ significantly between singular and combined forms, except for a slight variance in confidence interval of branches. As the phylogenetic trees provide more thorough and deeper conception of species relations, it is hoped that they would be useful to illuminate some forensic gaps in regions with high crime rates enriched by these plants, not only in Iran, but also in all areas over the world with this vegetation.

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

BACKGROUND: Anti-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. RESULTS: Bacterial 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. CONCLUSION: This 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.

The diversification of Caribbean Buxus in time and space: elevated speciation rates in lineages that accumulate nickel and spreading to other islands from Cuba in non-obligate ultramafic species.

BACKGROUND AND AIMS: The genus Buxus has high levels of endemism in the Caribbean flora, with ~50 taxa. In Cuba, 82 % grow on ultramafic substrates and 59 % are nickel (Ni) accumulators or Ni hyperaccumulators. Hence it is an ideal model group to study if this diversification could be related to adaptation to ultramafic substrates and to Ni hyperaccumulation. METHODS: We generated a well-resolved molecular phylogeny, including nearly all of the Neotropical and Caribbean Buxus taxa. To obtain robust divergence times we tested for the effects of different calibration scenarios, and we reconstructed ancestral areas and ancestral character states. Phylogenetic trees were examined for trait-independent shifts in diversification rates and we used multi-state models to test for state-dependent speciation and extinction rates. Storms could have contributed to Cuba acting as a species pump and to Buxus reaching other Caribbean islands and northern South America'. KEY RESULTS: We found a Caribbean Buxus clade with Mexican ancestors, encompassing three major subclades, which started to radiate during the middle Miocene (13.25 Mya). Other Caribbean islands and northern South America were reached from ~3 Mya onwards. CONCLUSIONS: An evolutionary scenario is evident in which Buxus plants able to grow on ultramafic substrates by exaptation became ultramafic substrate endemics and evolved stepwise from Ni tolerance through Ni accumulation to Ni hyperaccumulation, which has triggered species diversification of Buxus in Cuba. Storms could have contributed to Cuba acting as a species pump and to Buxus reaching other Caribbean islands and northern South America'.

The influence of climate and population density on Buxus hyrcana potential distribution and habitat connectivity.

Changes in environmental factors, human impact, and interactions between them accelerate the extinction of woody species. Therefore, conservation programs are needed to protect endangered taxa. However, the relationship between climate, habitat fragmentation, and anthropogenic activities and their consequences are still not well understood. In this work, we aimed to evaluate the impact of climate change and human population density on the Buxus hyrcana Pojark distribution range, as well as the phenomenon of habitat fragmentation. Based on species occurrence data throughout the Hyrcanian Forests (north of Iran), the MAXENT model was employed to estimate the potential distribution and suitability changes. Morphological-spatial analysis (MSPA) and CIRCUITSCAPE were used to assess habitat fragmentation and its connectivity. According to the main results obtained from future scenarios, the potential range will significantly decrease due to the lack of suitable climatic conditions. Meanwhile, B. hyrcana may not be able to shift in potentially suitable areas because of human influence and geographic barriers. Under RCP scenarios the extent of the core area would be reduced and the edge/core ratio significantly increased. Altogether, we found negative effects of the environmental change and the human population density on the continuity of habitats of B. hyrcana. The results of the presented work may improve our knowledge connected with in situ and ex situ protection strategies.

Ecophysiological responses of individually and group reared Cydalima perspectalis (Lepidoptera: Crambidae) to alkaloid-containing host plants.

The box tree moth, Cydalima perspectalis (Walker), is an invasive pest of Hyrcanian forests in Iran. In the current study, the effects of two boxwood species, Buxus hyrcana Pojark and B. microphylla Sieb. and Zucc. (Buxales: Buxaceae), were examined on ecophysiology of the pest. The demographic parameters were evaluated based on the age-stage, two-sex life table theory. The physiological characteristics of C. perspectalis were considered through fresh/dry weights and individually/group rearing methods. The results showed that C. perspectalis larvae were able to develop, survive and reproduce on both boxwood species. The developmental times were 41.62 and 41.46 days on B. hyrcana and B. microphylla, respectively. The total lifespan did not differ on boxwood species. However, the difference of fecundity on B. hyrcana (412.82 eggs/female) and B. microphylla (286.06 eggs/female) was significant. The intrinsic rate of increase (r) and finite rate of increase (λ) were significantly higher on B. hyrcana than B. microphylla. The nutritional indices showed that the consumption index (CI) on B. hyrcana were significantly lower than on B. microphylla based on fresh/dry weights in both rearing methods. Conversely, the efficiency of conversion of digested food (ECD) and the efficiency of conversion of ingested food (ECI) on B. hyrcana were significantly higher than on B. microphylla. Despite nonsignificant differences in leaf nutrient contents (except calcium), total protein, triglyceride, and alkaline phosphatase activity were inversely different in larvae compared to adult stage on boxwood species. These findings could help to comprehensively understand the performance of the pest on boxwood species.

Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

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.

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 United States. 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), B. sempervirens × B. microphylla var. koreana '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 to 13 days postinoculation (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 and 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-day 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.

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), an economically devastating disease affecting everyone in the supply chain from growers to gardeners, 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. Instead of abandoning boxwood production and switching to other evergreens, growers in the United States, informed by the latest research, have elected to combat this disease. One of the strategies employed has been to shift boxwood production from highly susceptible to less-susceptible cultivars. The objective of this study was to investigate the ongoing shift by comparing boxwood sales of 17 selected nurseries from seven states across the country in 2011, 2016, and 2021. Results revealed an additional 21.24% (from 38.58 to 60.04%) in sales of less-susceptible boxwood cultivars in 2021 compared with 2016. The less-susceptible cultivars responsible for increased boxwood sales are 'Winter Gem', 'Wintergreen', 'SB 300' (Freedom), 'SB 108' (Independence), and 'Little Missy'. The potential for long-term positive impact on sustainable boxwood production and plantings in the United States through the use of less-susceptible cultivars is discussed. This shift in boxwood choices builds crop health into new plantings of this landmark plant and sustains growth in demand for boxwood. This sets a new example of sustainable protection for a crop that is under serious pressure from an invasive pathogen.

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.

The current state of forest ecosystems in the Khosta Yew-Boxwood Grove.

In this study, we aimed to assess the current state of forest ecosystems on the Black Sea coast of the Caucasus after the mass mortality of boxwoods. Soil and geobotanical studies were carried out in the Khosta Yew-Boxwood Grove, a convenient proving ground for assessment of the consequences of Cydalima perspectalis expansion. Hierarchical cluster analysis (nearest-neighbour and Euclidean distance methods), One-way ANOVA, and correlation analysis (Euclidean distance matrices for standardised data) were applied to process the soil and vegetation data. An increase in the illumination of the lower forest tiers due to the Buxus colchica destruction resulted in an intensive growth of vegetation cover and the formation of a soddy horizon in soils. These processes contributed to the accumulation of organic matter and high biological activity of the soils. The number of Buxus colchica seedlings was negatively correlated with the vegetation coverage and the number of grass and shrub species, as well as with some biological parameters of the soils. The most intensive seed regeneration of Buxus colchica was observed in forest plots with high crown density in the upper tier, undeveloped vegetation cover, and soddy soil horizon.

Innovative Conversion of Pretreated Buxus sinica into High-Performance Biocomposites for Potential Use as Furniture Material.

Traditional wood-based panels are usually made from large-diameter trees and rely on adhesives for compactness, which negatively impacts the environment and human health. However, the widely distributed small-diameter shrubs are good raw materials for wood-based panels with abundant fibers, but are often under-exploited. This research reports the preparation of self-bonding biocomposites from Buxus sinica by an innovative combined approach of extraction, alkali treatment, and hot molding. The resulted biocomposites show better mechanical properties in which the flexural modulus (7.79 GPa) and the tensile modulus (4.33 GPa) were 5 times and 1.7 times higher than the conventional fiberboard, respectively, and also demonstrated better hydrophobicity than fiberboard, which could be due to the layer of lignin that formed on its surface preventing the infiltration of water. To sum up, the biocomposites prepared from small-diameter shrubs meet the requirement of the furniture and architectural decoration materials, suggesting that the proposed approach can be used to produce high-performance biocomposites.

A high-quality Buxus austro-yunnanensis (Buxales) genome provides new insights into karyotype evolution in early eudicots.

BACKGROUND: Eudicots are the most diverse group of flowering plants that compromise five well-defined lineages: core eudicots, Ranunculales, Proteales, Trochodendrales, and Buxales. However, the phylogenetic relationships between these five lineages and their chromosomal evolutions remain unclear, and a lack of high-quality genome analyses for Buxales has hindered many efforts to address this knowledge gap. RESULTS: Here, we present a high-quality chromosome-level genome of Buxus austro-yunnanensis (Buxales). Our phylogenomic analyses revealed that Buxales and Trochodendrales are genetically similar and classified as sisters. Additionally, both are sisters to the core eudicots, while Ranunculales was found to be the first lineage to diverge from these groups. Incomplete lineage sorting and hybridization were identified as the main contributors to phylogenetic discordance (34.33%) between the lineages. In fact, B. austro-yunnanensis underwent only one whole-genome duplication event, and collinear gene phylogeny analyses suggested that separate independent polyploidizations occurred in the five eudicot lineages. Using representative genomes from these five lineages, we reconstructed the ancestral eudicot karyotype (AEK) and generated a nearly gapless karyotype projection for each eudicot species. Within core eudicots, we recovered one common chromosome fusion event in asterids and malvids, respectively. Further, we also found that the previously reported fused AEKs in Aquilegia (Ranunculales) and Vitis (core eudicots) have different fusion positions, which indicates that these two species have different karyotype evolution histories. CONCLUSIONS: Based on our phylogenomic and karyotype evolution analyses, we revealed the likely relationships and evolutionary histories of early eudicots. Ultimately, our study expands genomic resources for early-diverging eudicots.

Comparative genomic analysis reveals contraction of gene families with putative roles in pathogenesis in the fungal boxwood pathogens Calonectria henricotiae and C. pseudonaviculata.

BACKGROUND: Boxwood blight disease caused by Calonectria henricotiae and C. pseudonaviculata is of ecological and economic significance in cultivated and native ecosystems worldwide. Prior research has focused on understanding the population genetic and genomic diversity of C. henricotiae and C. pseudonaviculata, but gene family evolution in the context of host adaptation, plant pathogenesis, and trophic lifestyle is poorly understood. This study applied bioinformatic and phylogenetic methods to examine gene family evolution in C. henricotiae, C. pseudonaviculata and 22 related fungi in the Nectriaceae that vary in pathogenic and saprobic (apathogenic) lifestyles. RESULTS: A total of 19,750 gene families were identified in the 24 genomes, of which 422 were rapidly evolving. Among the six Calonectria species, C. henricotiae and C. pseudonaviculata were the only species to experience high levels of rapid contraction of pathogenesis-related gene families (89% and 78%, respectively). In contrast, saprobic species Calonectria multiphialidica and C. naviculata, two of the closest known relatives of C. henricotiae and C. pseudonaviculata, showed rapid expansion of pathogenesis-related gene families. CONCLUSIONS: Our results provide novel insight into gene family evolution within C. henricotiae and C. pseudonaviculata and suggest gene family contraction may have contributed to limited host-range expansion of these pathogens within the plant family Buxaceae.

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.

Rapid Decline of Calonectria pseudonaviculata Soil Population in Selected Gardens Across the United States.

Calonectria pseudonaviculata (Cps) poses a serious threat to boxwood, an iconic landscape plant in American and European gardens. Under the mild climatic conditions of the United Kingdom, Cps remained recoverable in infected leaf debris after being left on the soil surface or buried for 5 years. The primary objective of this study was to determine how this fungus may be affected by the warmer summers and colder winters in the United States by sampling and baiting soil with boxwood cuttings and by on-site testing with sentinel plants. Soil sampling started in a Virginia garden in January 2016 and was extended to California, Illinois, New York, and South Carolina in early summer of 2017 through late fall of 2018. The Cps soil population as measured by the percentage of infected bait leaves declined sharply within the first year of blighted boxwood removal and fell to an almost undetectable level at the end of this study. To validate these baiting results, the Virginia garden was tested on site four times with container-grown boxwood plants while the South Carolina garden and three New York gardens were tested once. Each test began with sentinel plants set out for field exposure, followed by evaluation on site and then in the laboratory after plants were retrieved from these gardens and incubated under conducive environments for 2 weeks. Cps was not observed on any sentinel boxwood plant on site or in the laboratory with one exception. These observations indicate that Cps did not survive in the United States garden soil over time as well as it did in the United Kingdom. These results have important practical implications while challenging the notion that fungi producing microsclerotia will always survive in the soil for many years.

Buxus and Tetracentron genomes help resolve eudicot genome history.

Ancient whole-genome duplications (WGDs) characterize many large angiosperm lineages, including angiosperms themselves. Prominently, the core eudicot lineage accommodates 70% of all angiosperms and shares ancestral hexaploidy, termed gamma. Gamma arose via two WGDs that occurred early in eudicot history; however, the relative timing of these is unclear, largely due to the lack of high-quality genomes among early-diverging eudicots. Here, we provide complete genomes for Buxus sinica (Buxales) and Tetracentron sinense (Trochodendrales), representing the lineages most closely related to core eudicots. We show that Buxus and Tetracentron are both characterized by independent WGDs, resolve relationships among early-diverging eudicots and their respective genomes, and use the RACCROCHE pipeline to reconstruct ancestral genome structure at three key phylogenetic nodes of eudicot diversification. Our reconstructions indicate genome structure remained relatively stable during early eudicot diversification, and reject hypotheses of gamma arising via inter-lineage hybridization between ancestral eudicot lineages, involving, instead, only stem lineage core eudicot ancestors.