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.

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.

Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata.

Pathogen detection and identification are key elements in outbreak control of human, animal, and plant diseases. Since many fungal plant pathogens cause similar symptoms, are difficult to distinguish morphologically, and grow slowly in culture, culture-independent, sequence-based diagnostic methods are desirable. Whole genome metagenomic sequencing has emerged as a promising technique because it can potentially detect any pathogen without culturing and without the need for pathogen-specific probes. However, efficient DNA extraction protocols, computational tools, and sequence databases are required. Here we applied metagenomic sequencing with the Oxford Nanopore Technologies MinION to the detection of the fungus Calonectria pseudonaviculata, the causal agent of boxwood (Buxus spp.) blight disease. Two DNA extraction protocols, several DNA purification kits, and various computational tools were tested. All DNA extraction methods and purification kits provided sufficient quantity and quality of DNA. Several bioinformatics tools for taxonomic identification were found suitable to assign sequencing reads to the pathogen with an extremely low false positive rate. Over 9% of total reads were identified as C. pseudonaviculata in a severely diseased sample and identification at strain-level resolution was approached as the number of sequencing reads was increased. We discuss how metagenomic sequencing could be implemented in routine plant disease diagnostics.

Evidence for the Role of CYP51A and Xenobiotic Detoxification in Differential Sensitivity to Azole Fungicides in Boxwood Blight Pathogens.

Boxwood blight, a fungal disease of ornamental plants (Buxus spp.), is caused by two sister species, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Compared to Cps, Che is documented to display reduced sensitivity to fungicides, including the azole class of antifungals, which block synthesis of a key fungal membrane component, ergosterol. A previous study reported an ergosterol biosynthesis gene in Cps, CYP51A, to be a pseudogene, and RNA-Seq data confirm that a functional CYP51A is expressed only in Che. The lack of additional ergosterol biosynthesis genes showing significant differential expression suggests that the functional CYP51A in Che could contribute to reduced azole sensitivity when compared to Cps. RNA-Seq and bioinformatic analyses found that following azole treatment, 55 genes in Cps, belonging to diverse pathways, displayed a significant decrease in expression. Putative xenobiotic detoxification genes overexpressed in tetraconazole-treated Che encoded predicted monooxygenase and oxidoreductase enzymes. In summary, expression of a functional CYP51A gene and overexpression of predicted xenobiotic detoxification genes appear likely to contribute to differential fungicide sensitivity in these two sister taxa.

Comparative Performance of Sanitizers in Managing Plant-to-Plant Transfer and Postharvest Infection of Calonectria pseudonaviculata and Pseudonectria foliicola on Boxwood.

Calonectria pseudonaviculata and Pseudonectria foliicola causing the infamous "boxwood blight" and "Volutella blight," respectively, are a constant threat to the boxwood production and cut boxwood greenery market. Both pathogens cause significant economic loss to all parties (growers, retailer, and customers) in the horticultural chain. The objective of this study was to evaluate efficacy of disinfesting chemicals (quaternary ammonium compound [QAC], peroxy, acid, alcohol, chlorine, and cleaner) in preventing plant-to-plant transfer of C. pseudonaviculata and P. foliicola via cutting tools, as well as reduction of postharvest boxwood blight and Volutella blight disease severity in harvested boxwood greenery. First, an in vitro study was conducted to select products and doses that completely or near-completely inhibited conidial germination of C. pseudonaviculata and P. foliicola. The selected treatments were also tested for their ability to reduce plant-to-plant transfer of C. pseudonaviculata and P. foliicola and manage postharvest boxwood blight and Volutella blight in boxwood cuttings. For the plant-to-plant transfer study, Felco 19 shears were used as a tool for mechanical transfer of fungal conidia. The blades of Felco 19 shears were exposed to a conidial suspension of C. pseudonaviculata or P. foliicola by cutting a 1-cm-diameter cotton roll that had been dipped into a fungal suspension. Disease-free boxwood rooted cuttings (10-cm height) were pruned with the contaminated shears. The Felco 19 shears were equipped with a mounted miniature sprayer connected to a pressurized reservoir of treatment solution that automatically sprayed the blade and plant surface while cutting. The influence of accumulated sap on the shear blade was studied through 1- or 10-cut pruning variable on test plants and screened for the efficacy of treatments. Then, the boxwood rooted cuttings were transplanted and incubated in room conditions (21°C, 60% RH) with 12 h of fluorescent light; data evaluation on disease severity was done weekly for a month. Disease progress (area under disease progress curve [AUDPC]) was calculated. In another study, postharvest dip application treatments were used for the management of postharvest boxwood blight or Volutella blight on boxwood cuttings. The harvested boxwood cuttings were inoculated with a conidial suspension of C. pseudonaviculata or P. foliicola and then dipped into treatment solution 3 days afterward. The treated boxwood cuttings were kept in room conditions, and boxwood blight or Volutella blight disease severity as well as marketability (postharvest shelf life) was assessed every 2 days for 1 week. A significant difference between treatments was observed for reduction of boxwood blight or Volutella blight severity and AUDPC. The treatments [Octyl decyl dimethyl (ODD) + dioctyl dimethyl (DoD) + didecyl dimethyl (DdD) + dimethyl benzyl (DB)] ammonium chloride (AC) (Simple Green D Pro 5), 2-propanol + didecyl dimethyl ammonium chloride (DDAC) (0.12%; KleenGrow), and dimethyl benzyl ammonium chloride (DBAC) + dimethyl ethylbenzyl ammonium chloride (DEAC) (GreenShield) were the most effective in reducing the plant-to-plant transfer of boxwood blight and Volutella blight when pruned with contaminated Felco 19 shears. In addition to the three effective treatments above, acetic acid (2.5%; vinegar), 2-propanol + DDAC (0.06%), sodium hypochlorite (Clorox), and potassium peroxymonosulfate + NaCl (2%; Virkon) were effective in reducing postharvest boxwood blight, whereas DBAC + DBAC (Lysol all-purpose cleaner), ethanol (70% [ethyl alcohol]), and DDAC + DBAC (Simple Green D Pro 3 plus) were effective in reducing Volutella blight disease severity and AUDPC, and they also maintained better quality and longer postharvest shelf life of boxwood cuttings when applied as a dip treatment. The longer postharvest shelf life of boxwood cuttings noted may be attributed to reduced disease severity and AUDPC resulting in healthy boxwood cuttings.

Global distribution of mating types shows limited opportunities for mating across populations of fungi causing boxwood blight disease.

Boxwood blight is a disease threat to natural and managed landscapes worldwide. To determine mating potential of the fungi responsible for the disease, Calonectria pseudonaviculata and C. henricotiae, we characterized their mating-type (MAT) loci. Genomes of C. henricotiae, C. pseudonaviculata and two other Calonectria species (C. leucothoes, C. naviculata) were sequenced and used to design PCR tests for mating-type from 268 isolates collected from four continents. All four Calonectria species have a MAT locus that is structurally consistent with the organization found in heterothallic ascomycetes, with just one idiomorph per individual isolate. Mating type was subdivided by species: all C. henricotiae isolates possessed the MAT1-1 idiomorph, whereas all C. pseudonaviculata isolates possessed the MAT1-2 idiomorph. To determine the potential for divergence at the MAT1 locus to present a barrier to interspecific hybridization, evolutionary analysis was conducted. Phylogenomic estimates showed that C. henricotiae and C. pseudonaviculata diverged approximately 2.1 Mya. However, syntenic comparisons, phylogenetic analyses, and estimates of nucleotide divergence across the MAT1 locus and proximal genes identified minimal divergence in this region of the genome. These results show that in North America and parts of Europe, where only C. pseudonaviculata resides, mating is constrained by the absence of MAT1-1. In regions of Europe where C. henricotiae and C. pseudonaviculata currently share the same host and geographic range, it remains to be determined whether or not these two recently diverged species are able to overcome species barriers to mate.

Host responses and impact on the boxwood blight pathogen, Calonectria pseudonaviculata.

MAIN CONCLUSION: Boxwood leaves are more susceptible to Calonectria pseudonaviculata (Cps) and better suited for Cps reproduction than those of pachysandra and sweet box. Passages through a non-boxwood host may alter Cps ability to sporulate. Calonectria pseudonaviculata (Cps) infects boxwood and its two common companion plants-pachysandra and sweet box. This study investigated how boxwood, pachysandra, and sweet box respond to Cps isolates of different host origin. Detached leaves were inoculated with nine isolates, three from each host, and evaluated for colonization, infection rate, lesion size, and production of conidia and microsclerotia. Cps colonized boxwood leaf tissue within 12 h of inoculation, and 60 h ahead of pachysandra and sweet box. Cps also produced significantly larger lesions and more conidia on boxwood than on pachysandra and sweet box. Isolates originating from different host plants did not differ in all the components evaluated except for conidia production. Isolates from boxwood and sweet box produced significantly more conidia than those from pachysandra. Overall, boxwood leaves are more susceptible to the disease and are better suited for Cps reproduction than those of pachysandra and sweet box. Passages through a non-boxwood host may alter Cps ability to sporulate. These results advance the understanding of Cps biology and affirm the importance of taking pachysandra and sweet box into consideration in disease management planning.

Preventing Soil Inoculum of Calonectria pseudonaviculata from Splashing onto Healthy Boxwood Foliage by Mulching.

Boxwood blight, caused by Calonectria pseudonaviculata, is an emerging disease of great concern to horticulturists in the United States and other affected countries. The objective of this study was to evaluate the efficacy of mulching as a physical barrier to prevent soil inoculum from splashing onto healthy boxwood foliage. A field trial consisting of two treatments, mulched and nonmulched, was conducted under field conditions in Lowgap, North Carolina, and in a residential landscape setting near Richmond, Virginia, for 2 years at each site. Mulching efficacy was assessed by monitoring and comparing boxwood blight development on detector plants: containerized 'Justin Brouwers' boxwood, which were rotated through mulched and nonmulched plots at 1- and 2-week intervals in the Lowgap and Richmond sites, respectively. Boxwood blight was observed on detector plants in a combined 55 of the 88 monitoring periods during this study at the two sites. Mulching provided complete protection of Justin Brouwers boxwood from infection by C. pseudonaviculata soil inoculum during 33 of the 55 positive monitoring periods (60%) and good to excellent protection during 13 monitoring periods (24%). The potential applications of mulching for boxwood blight mitigation are discussed.

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.

Boxwood blight: an ongoing threat to ornamental and native boxwood.

Boxwood blight is an emerging disease of ornamental and native boxwood plants in the family Buxaceae. First documented in the 1990s at a single location in England, the disease is now reported throughout Europe, Asia, New Zealand, and North America. To address the growing concern over boxwood blight, ongoing research focuses on multiple biological and genetic aspects of the causal pathogens and susceptible host plants. Characterization of genetic variation among the Calonectria fungi that cause boxwood blight shows that two unique sister species with different geographic distributions incite the disease. Studies of the pathogen life cycle show the formation of long-lived survival structures and that host infection is dependent on inoculum density, temperature, and humidity. Host range investigations detail high levels of susceptibility among boxwood as well as the potential for asymptomatic boxwood infection and for other plants in the family Buxaceae to serve as additional hosts. Multiple DNA-based diagnostic assays are available, ranging from probe-based quantitative PCR assays to the use of comparative genomics to develop robust diagnostic markers or provide whole genome-scale identifications. Though many questions remain, the research that continues to address boxwood blight demonstrates the importance of applying a multidisciplinary approach to understand and control emerging plant diseases.

Microsclerotial Enumeration, Size, and Survival of Calonectria pseudonaviculata.

Boxwood blight caused by Calonectria pseudonaviculata (Cps) is an emerging disease in the United States. Understanding the biology and survival of microsclerotia, the resting propagules of Cps, is important to managing the disease and restoring Cps-infested sites. In this study, an image-analyzing software (OpenCFU) was adapted to enumerate microsclerotia produced in culture plates. Microsclerotial counts increased with time up to 42 days while their size continued to grow for 70 days. Additionally, younger microsclerotia were less sensitive to extreme temperatures of -10 and 40°C than older ones. For instance, 21-day-old microsclerotia survived at almost twice the rate of 70-day-old ones after a 24-h exposure to -10°C. Likewise, the 21-day-old microsclerotia survived at much greater rates than 70-day-old ones (50 to 70% versus <10%) after a 24-h exposure to 40°C. The practical implications of these results are discussed.

LAMP Detection Assays for Boxwood Blight Pathogens: A Comparative Genomics Approach.

Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.

Antibacterial Azaphilones from an Endophytic Fungus, Colletotrichum sp. BS4.

Three new compounds, colletotrichones A-C (1-3), and one known compound, chermesinone B (4a), were isolated from an endophytic fungus, Colletotrichum sp. BS4, harbored in the leaves of Buxus sinica, a well-known boxwood plant used in traditional Chinese medicine (TCM). Their structures were determined by extensive spectroscopic analyses including 1D and 2D NMR, HRMS, ECD spectra, UV, and IR, as well as single-crystal X-ray diffraction, and shown to be azaphilones sharing a 3,6a-dimethyl-9-(2-methylbutanoyl)-9H-furo[2,3-h]isochromene-6,8-dione scaffold. Owing to the remarkable antibacterial potency of known azaphilones coupled to the usage of the host plant in TCM, we evaluated the antibacterial efficacy of the isolated compounds against two commonly dispersed environmental strains of Escherichia coli and Bacillus subtilis, as well as against two human pathogenic clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa. Compound 1 exhibited marked antibacterial potencies against the environmental strains that were comparable to the standard antibiotics. Compound 3 was also active against E. coli. Finally, compound 2a exhibited the same efficacy as streptomycin against the clinically relevant bacterium S. aureus. The in vitro cytotoxicity of these compounds on a human acute monocytic leukemia cell line (THP-1) was also assessed. Our results provide a scientific rationale for further investigations into endophyte-mediated host chemical defense against specialist and generalist pathogens.

A new species of Meliolinites associated with Buxus leaves from the Oligocene of Guangxi, southern China.

A new species of Meliolinites (fossil Meliolaceae), M. buxi sp. nov., is reported from the Oligocene Ningming Formation of Guangxi, South China. The fungus has hyphopodia characteristics of extant Meliolaceae, such as thick-walled, branching hyphae with appressoria and phialides. However, these fossils entirely lack mycelial or perithecial setae and have only a few phialides, thereby distinguishing the new species from most known species. The fungus was discovered on the adaxial and abaxial cuticles of several fossilized Buxus leaves. Thickening and twisting of cell walls in the Buxus leaf cuticle, along with the parasitic feeding strategy of the extant Meliolaceae, suggest that a parasitic interaction between Buxus and M. buxi seems feasible. The distribution of modern Meliolaceae suggests that they live in warm, humid subtropical-tropical climates. It is possible that the presence of M. buxi indicates a similar climatic condition. The co-occurrence of large-leaf Buxus and floristic comparisons of the Ningming assemblage also corroborate this conclusion.