Complete Genome Sequence Resource for Xanthomonas hortorum Isolated from Greek Oregano.

In spring 2019, necrotic leaf spots were detected on Greek oregano (Origanum vulgare var. hirtum) plants in a commercial greenhouse operation. An isolate was recovered from the diseased plants. Partial 16S ribosomal RNA sequencing and multilocus sequence analysis revealed that the isolate was a Xanthomonas sp. but proved insufficient to identify the species with certainty. Therefore, whole-genome sequencing using both Nanopore and Illumina technologies was performed. Here, we report the complete and annotated genome sequence of Xanthomonas hortorum strain 108, which was originally isolated from Greek oregano in Long Island, NY, U.S.A.

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.

Genomic insights into a Pseudomonas amygdali isolate from Hibiscus rosa-sinensis.

The taxonomy of Pseudomonas has been extensively studied, yet the determination of species is currently difficult because of recent taxonomic changes and the lack of complete genomic sequence data. We isolated a bacterium causing a leaf spot disease on hibiscus (Hibiscus rosa-sinensis). Whole genome sequencing revealed similarity to Pseudomonas amygdali pv. tabaci and pv. lachrymans. The genome of this isolate (referred to as P. amygdali 35-1) shared 4987 genes with P. amygdali pv. hibisci, but possessed 204 unique genes and contained gene clusters encoding putative secondary metabolites and copper resistance determinants. We predicted this isolate's type III secretion effector (T3SE) repertoire and identified 64 putative T3SEs, some of which are present in other P. amygdali pv. hibisci strains. Assays showed that the isolate was resistant to copper at a concentration of 1.6 mM. This study provides an improved understanding of the genomic relatedness and diversity of the P. amygdali species.

Characterization of Boxwood Shoot Bacterial Communities and Potential Impact from Fungicide Treatments.

Phyllosphere bacterial communities play important roles in plant fitness and growth. The objective of this study was to characterize the epiphytic and endophytic bacterial communities of boxwood shoots and determine how they may respond to commonly used fungicides. In early summer and early fall, shoot samples were collected immediately before and 1, 7, and 14 days after three fungicides containing chlorothalonil and/or propiconazole were applied to the canopy. Total genomic DNA from shoot surface washings and surface-sterilized shoot tissues was used as the template for 16S rRNA metabarcoding, and the amplicons were sequenced on a Nanopore MinION sequencer to characterize the epiphytic and endophytic communities. The bacterial communities were phylogenetically more diverse on the boxwood shoot surface than in the internal tissue, although the two communities shared 12.7% of the total 1,649 identified genera. The most abundant epiphytes were Methylobacterium and Pantoea, while Stenotrophomonas and Brevundimonas were the dominant endophytes. Fungicide treatments had strong impacts on epiphytic bacterial community structure and composition. Analysis of compositions of microbiomes with bias correction (ANCOM-BC) and analysis of variance (ANOVA)-like differential expression (ALDEx2) together identified 312 and 1,362 epiphytes changed in abundance due to fungicide treatments in early summer and early fall, respectively, and over 50% of these epiphytes were negatively impacted by fungicide. The two chlorothalonil-based contact fungicides demonstrated more marked effects than the propiconazole-based systemic fungicide. These results are foundational for exploring and utilizing the full potential of the microbiome and fungicide applications and developing a systems approach to boxwood health and production. IMPORTANCE Agrochemicals are important tools for safeguarding plants from invasive pathogens, insects, mites, and weeds. How they may affect the plant microbiome, a critical component of crop health and production, was poorly understood. Here, we used boxwood, an iconic low-maintenance landscape plant, to characterize shoot epiphytic and endophytic bacterial communities and their responses to contact and systemic fungicides. This study expanded our understanding of the above-ground microbiome in ornamental plants and is foundational for utilizing the full benefits of the microbiome in concert with different fungicide chemistries to improve boxwood health. This study also sets an example for a more thorough evaluation of these and other agrochemicals for their effects on boxwood microbiomes during production and offers an expanded systems approach that could be used with other crops for enhanced integrated pest management.

Differential Adaptation Has Resulted in Aggressiveness Variation of Calonectria pseudonaviculata on Hosts Buxus, Pachysandra, and Sarcococca.

Calonectria pseudonaviculata (Cps) infects Buxus (boxwood), Pachysandra (pachysandra), and Sarcococca spp. (sweet box); yet, how it adapts to its hosts has been unclear. Here, we performed serial passage experiments with the three hosts and measured Cps changes in three aggressiveness components: infectibility, lesion size, and conidial production. The detached leaves of individual hosts were inoculated with isolates (P0) from the originating host, followed by nine serial inoculations of new leaves of the same host with conidia from the infected leaves of the previous inoculation. All boxwood isolates maintained their capability of infection and lesion expansion through the 10 passages, whereas most non-boxwood isolates lost these abilities during the passages. Isolates from plants of origin (*-P0) and their descendants isolated from passages 5 (*-P5) and 10 (*-P10) were used to evaluate aggressiveness changes on all three hosts with cross-inoculation. While post-passage boxwood isolates gave enlarged lesions on pachysandra, sweet box P5 and pachysandra P10 isolates showed reduced aggressiveness on all hosts. Cps appears to be most adapted to boxwood and less adapted to sweet box and pachysandra. These results suggest speciation of Cps, with its coevolutionary pace with the hosts the fastest with boxwood, intermediate with sweet box, and the slowest with pachysandra.

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.

Whole Genome Sequencing-Based Tracing of a 2022 Introduction and Outbreak of Xanthomonas hortorum pv. pelargonii.

Globalization has made agricultural commodities more accessible, available, and affordable. However, their global movement increases the potential for invasion by pathogens and necessitates development and implementation of sensitive, rapid, and scalable surveillance methods. Here, we used 35 strains, isolated by multiple diagnostic laboratories, as a case study for using whole genome sequence data in a plant disease diagnostic setting. Twenty-seven of the strains were isolated in 2022 and identified as Xanthomonas hortorum pv. pelargonii. Eighteen of these strains originated from material sold by a plant breeding company that had notified clients following a release of infected geranium cuttings. Analyses of whole genome sequences revealed epidemiological links among the 27 strains from different growers that confirmed a common source of the outbreak and uncovered likely secondary spread events within facilities that housed plants originating from different plant breeding companies. Whole genome sequencing data were also analyzed to reveal how preparatory and analytical methods can impact conclusions on outbreaks of clonal pathogenic strains. The results demonstrate the potential power of using whole genome sequencing among a network of diagnostic labs and highlight how sharing such data can help shorten response times to mitigate outbreaks more expediently and precisely than standard methods.

Identification and Characterization of Pythium, Globisporangium, and Phytopythium Species Present in Floricultural Crops from Long Island, New York.

Several Pythium, Globisporangium, and Phytopythium species cause Pythium diseases in greenhouse floricultural crops, resulting in significant seasonal losses. Four hundred and eighteen Pythium, Globisporangium, and Phytopythium isolates from flowering crops, growing media, or bench and floor debris were collected from Long Island greenhouses or clinic samples between 2002 and 2013. Isolates were identified to species based on morphology and internal transcribed spacer barcoding. Twenty-two species of Pythium, Phytopythium, and Globisporangium were identified, with Globisporangium irregulare sensu lato (s.l.) being the most common. To determine the origin of inoculum during the 2011 cropping season, 11 microsatellite loci were analyzed in 124 G. irregulare s.l. isolates collected in four greenhouses and six previously collected from clinic samples. Cluster analyses grouped G. irregulare s.l. isolates into four groups: G. irregulare sensu stricto, plus three G. cryptoirregulare clusters. The population structure defined by greenhouse and host was found in two clades. Additionally, the population dynamics of G. irregulare s.l. isolates associated with Pelargonium spp. from 2011 to 2013 were examined using 85 isolates and nine informative microsatellite loci to assess inoculum survival over multiple cropping seasons. Although most isolates had unique genotypes, closely related genotypes were found in the same locations over different years. Our results indicate that G. irregulare s.l. inocula have local as well as remote origins. Isolates may be initially brought into ornamental operations from common sources, such as infected plant materials or infested potting mixes. Our results support the hypothesis that established strains can serve as inocula and survive in greenhouse facilities over multiple seasons.

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.

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.

Characterization of the Soil Bacterial Community from Selected Boxwood Gardens across the United States.

In a recent study, we observed a rapid decline of the boxwood blight pathogen Calonectria pseudonaviculata (Cps) soil population in all surveyed gardens across the United States, and we speculated that these garden soils might be suppressive to Cps. This study aimed to characterize the soil bacterial community in these boxwood gardens. Soil samples were taken from one garden in California, Illinois, South Carolina, and Virginia and two in New York in early summer and late fall of 2017 and 2018. Soil DNA was extracted and its 16S rRNA amplicons were sequenced using the Nanopore MinION® platform. These garden soils were consistently dominated by Rhizobiales and Burkholderiales, regardless of garden location and sampling time. These two orders contain many species or strains capable of pathogen suppression and plant fitness improvement. Overall, 66 bacterial taxa were identified in this study that are known to have strains with biological control activity (BCA) against plant pathogens. Among the most abundant were Pseudomonas spp. and Bacillus spp., which may have contributed to the Cps decline in these garden soils. This study highlights the importance of soil microorganisms in plant health and provides a new perspective on garden disease management using the soil microbiome.

Genomic characterization of a new torradovirus from common fleabane (Erigeron annuus).

A new virus was detected in common fleabane (Erigeron annuus) showing virus-like symptoms including leaf yellowing, mosaic, and mottling. This virus is tentatively named "fleabane yellow mosaic virus" (FbYMV). The complete genome sequence consists of two RNA segments of 7,133 nt (RNA 1) and 4,810 nt (RNA 2), excluding the poly(A) tract. Sequence analysis showed a genome organization comparable to that of members of the genus Torradovirus. The level of sequence identity between FbYMV and known members of the genus Torradovirus was below the cutoff established by the ICTV for species demarcation. Therefore, FbYMV should be classified as a new member of the genus Torradovirus.

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.

First Report of Dickeya dianthicola causing blackleg on New Guinea Impatiens (Impatiens hawkeri) in New York State, USA.

New Guinea impatiens (NGI), Impatiens hawkeri, has a $54-million wholesale market value in the United States (National Agricultural Statistics Service, 2019) and is highly resistant to Impatiens downy mildew (Plasmopara obducens) according to growers' experience (Warfield, 2011). In March 2019, NGI cv. Petticoat White in a New York greenhouse showed wilting, black stem streaks and vascular discoloration, with a 20% disease incidence. Symptomatic tissue pieces were added to sterile water in a test tube and streaks made on potato dextrose agar (PDA). After incubation at 26oC for two days, the most abundant colony type (mucoid, pale yellow) was transferred to PDA. One representative colony was selected and labeled as isolate 67-19. A single colony of isolate 67-19 was transferred to lysogeny broth (LB) (Bertani, 1951) and cultured at 28oC. Genomic DNA was extracted and polymerase chain reaction (PCR) performed using the 16S rRNA gene universal primers fD2 and rP1 resulting in a partial 16S rRNA amplicon (Weisburg et al., 1991). Basic Local Alignment Search Tool (BLASTn) analysis (Altschul et al., 1990) showed 99% identity with sequences of species belonging to Dickeya. Different primer sets have been developed to detect and identify the genus Dickeya and its various species (Pritchard et al., 2013). The primer sets used for genus identification, dnaX (Slawiak et al., 2009), Df/Dr (Laurila et al., 2010) and ADE1/ADE2 (Nassar et al., 1996), resulted in 500-bp, 133-bp, and 420-bp amplicons, respectively. Results suggested the bacterium was a Dickeya sp. To determine whether the species could be D. dianthicola, the specific primer set DIA-A was used (Pritchard et al., 2013) and the expected product of 150-bp was obtained. BLASTn results showed that the partial dnaX sequence (GenBank accession MT895847) of isolate 67-19 had 99% identity with the sequence of D. dianthicola strain RNS04.9 isolated in 2004 from potato (Solanum tuberosum) in France (GenBank accession CP017638.1). Therefore, this isolate 67-19 was designated as D. dianthicola. The complete genome of D. dianthicola strain 67-19 was generated using Nanopore and Illumina sequencing (GenBank accession CP051429) (Liu et al., 2020). Average nucleotide identity (ANI) determined by FastANI (v1.1) (Jain et al., 2018) showed 97.43% identity between the genome of D. dianthicola strain 67-19 and that of D. dianthicola strain NCPPB 453 (GenBank accession GCA_000365305.1), isolated in 1957 from carnation (Dianthus caryophyllus) in the UK. The pathogenicity of D. dianthicola strain 67-19 was shown on NGI cultivars Petticoat White and Tamarinda White. In July 2020, sterile toothpicks were used to make wounds and to transfer bacteria from a 48-hr PDA culture of D. dianthicola strain 67-19 to the stems of four plants of each cultivar. Four plants of each cultivar were mock inoculated similarly and all wound sites were wrapped with Parafilm before placing plants on a greenhouse bench. Ten days later, stems inoculated with D. dianthicola strain 67-19 showed necrotic lesions similar to the original symptoms, while control plants did not show symptoms. One month after inoculation, bacteria were re-isolated from all symptomatic stems. PCR was performed on the re-isolated bacteria as described. The dnaX sequence (GenBank accession MT895847) was confirmed to match that of D. dianthicola strain 67-19 (GenBank accession CP051429) 100% and fragments of the expected size were amplified (Liu et al., 2020). Stab inoculations of strain 67-19 into potato stems and tubers also resulted in blackleg and soft rot symptoms at the sites of inoculation, while mock-inoculated stem and tuber showed no symptoms. The sequence of the dnaX gene of the re-isolated bacterium from inoculated potatoes was confirmed to match that of D. dianthicola strain 67-19. To our knowledge, this is the first report of blackleg of New Guinea impatiens caused by D. dianthicola in the United States and worldwide. Since the disease caused by D. dianthicola poses a significant threat to the ornamentals and potato industries (Charkowski et al., 2020), further research on genome biology, epidemiology and management options is needed. LITERATURE CITED Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403-410. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. Journal of Bacteriology 62:293-300. Charkowski, A., Sharma, K., Parker, M.L., Secor, G.A., and Elphinstone, J. 2020. Bacterial diseases of potato. Pages 351-388 in: The Potato Crop: Its Agricultural, Nutritional and Social Contribution to Humankind, H. Campos and O. Ortiz, eds. Springer International Publishing, Cham. Jain, C., Rodriguez-R, L.M., Phillippy, A.M., Konstantinidis, K.T., and Aluru, S. 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nature Communications 9:5114. Laurila, J., Hannukkala, A., Nykyri, J., Pasanen, M., Hélias, V., Garlant, L., and Pirhonen, M. 2010. Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland. European Journal of Plant Pathology 126:249-262. Liu, Y., Helmann, T., Stodghill, P., and Filiatrault, M. 2020. Complete genome sequence resource for the necrotrophic plant-pathogenic bacterium Dickeya dianthicola 67-19 isolated from New Guinea Impatiens. Plant Disease. https://doi.org/10.1094/PDIS-09-20-1968-A. Nassar, A., Darrasse, A., Lemattre, M., Kotoujansky, A., Dervin, C., Vedel, R., and Bertheau, Y. 1996. Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes. Applied and Environmental Microbiology 62:2228-2235. National Agricultural Statistics Service. 2019. Floriculture crops 2018 summary. ISSN: 1949-0917. https://downloads.usda.library.cornell.edu/usda-esmis/files/0p0966899/rr1728124/76537c134/floran19.pdf Pritchard, L., Humphris, S., Saddler, G.S., Parkinson, N.M., Bertrand, V., Elphinstone, J.G., and Toth, I.K. 2013. Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathology 62:587-596. Slawiak, M., van Beckhoven, J.R.C.M., Speksnijder, A.G.C.L., Czajkowski, R., Grabe, G., and van der Wolf, J.M. 2009. Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. European Journal of Plant Pathology 125:245-261. Warfield, C.Y. (2011). Downy Mildew of Impatiens. In GrowerTalks. https://www.growertalks.com/Article/?articleid=18921 Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173:697-703.

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.

Phlox Species Show Quantitative and Qualitative Resistance to a Population of Powdery Mildew Isolates from the Eastern United States.

Ornamental plants in the genus Phlox are extensively planted in landscapes and home gardens around the world. A major limitation to a more widespread use of these plants is their susceptibility to powdery mildew (PM). In this study, we used multilocus sequence typing (MLST) analysis to gain insights into the population diversity of 32 Phlox PM pathogen (Golovinomyces magnicellulatus and Podosphaera sp.) isolates collected from the eastern United States and relate it to the ability to overcome host resistance. Low genetic diversity and a lack of structure were found within our population. Whole genome comparison of two isolates was used to support low genetic diversity evidence found with the MLST analysis. Recombination was suggested by the incongruences observed in the six phylogenetic trees generated from the housekeeping genes TEF-1α, CSI, ITS, IGS, H3, and TUB. Contrasting with low genetic diversity, we found high phenotypic diversity when using 10 of the 32 isolates to evaluate host resistance in four different Phlox species (P. paniculata 'Dunbar Creek', P. amoena OPGC 3598, P. glaberrima OPGC 3594, and P. subulata OPGC 4185) using in vitro bioassays. We observed quantitative and qualitative resistance in all Phlox species and a consistent low disease severity in our control, P. paniculata 'Dunbar Creek'. Taken together, the results generated in this study constitute a robust screening of popular Phlox germplasm that can be incorporated into breeding programs for PM resistance and provides significant information on the evolution of PM pathogens.

Indications of Susceptibility to Calonectria pseudonaviculata in Some Common Groundcovers and Boxwood Companion Plants.

Knowing the host range of a pathogen is critical to developing and implementing effective disease management programs. Calonectria pseudonaviculata (Cps) is known to attack a number of species, varieties, and cultivars in the genus Buxus as well as three Pachysandra species (Pachysandra terminalis, Pachysandra procumbens, and Pachysandra axillaris) and several Sarcococca species, all in the Buxaceae family. The objective of this study was to evaluate non-Buxaceae groundcovers and companion plants commonly associated with boxwood plantings for their susceptibility to Cps. Twenty-seven plant species belonging to 21 families were exposed to different levels of inoculum: 50 to 300 conidia per drop for detached leaf assays and 30,000 to 120,000 conidia per 1 ml for whole-plant assays. Inoculated plants were incubated in humid environments for at least 48 h to facilitate infection. Cps infection and sporulation were observed on 12 plant species: Alchemilla mollis, Arctostaphylos uva-ursi, Brunnera macrophylla, Epimedium × youngianum, Galium odoratum, Geranium sanguineum, Phlox subulata, Tiarella cordifolia, Callirhoe involucrata, Iberis sempervirens, Mazus reptans, and Vinca minor. These results suggest that there may be more hosts of Cps commonly grown in nurseries and landscapes. If corroborated by observations of natural infection, these findings have implications for the Boxwood Blight Cleanliness Program instituted by the National Plant Board and for planning disease mitigation at production and in the landscape.

Boxwood Blight: Threat to Ornamentals.

Boxwood blight, caused by Calonectria pseudonaviculata and Calonectria henricotiae, has had devastating effects in gardens since its first appearance in the United Kingdom in 1994. The disease affects two other plants in the Buxaceae: sweet box (Sarcococca spp.) and pachysandra (Pachysandra spp.). C. pseudonaviculata was likely introduced to Europe by nursery trade from East Asia on an ornamental species and then to western Asia and North America. Thus far, C. henricotiae has been seen only in Europe. Boxwood, valued at $126 million wholesale per year in the United States alone, is now besieged by an aggressive foliar blight active over a broad temperature range when there are long periods of leaf wetness. Research on inoculum, means of dissemination, cultivar susceptibility, environmental influences, fungicides, sanitizers, and detection methods has vastly improved knowledge of this new invasive disease in a short time. Boxwood with genetic resistance to the disease is critically needed.

Downy Mildew: A Serious Disease Threat to Rose Health Worldwide.

Peronospora sparsa is a downy mildew-causing oomycete that can infect roses, blackberries, and other members of the rose family. During the last 70 years, this disease has become a serious problem for rose growers in the U.S. and worldwide. While much is known about the disease and its treatment, including significant research on molecular identification methods, as well as environmental conditions conducive to disease and the fungicides used to prevent it, significant knowledge gaps remain in our basic comprehension of the pathogen's biology. For example, the degree of genetic relatedness of pathogen isolates collected from rose, caneberries, and cherry laurel has never been examined, and the natural movement of genotypes from host to host is not known. Further work could be done to determine the differences in pathogen population structure over time (using herbarium specimens and fresh collections) or differences in pathogen population structure and pathogen environmental adaptation for specimens from different geographic regions. The oospore stage of the organism is poorly understood, both as to how it forms and whether it serves as an overwintering structure in nurseries and landscapes. In production greenhouses, the detection of the pathogen using infrared thermographic imaging and possible inhibition by ultraviolet light needs to be explored. Further work needs to be done on breeding using wild roses as new sources for resistance and using new methods such as marker assisted selection and RNAi technologies. As roses are one of the most economically important ornamental crops worldwide, a proper understanding of the disease cycle could allow for better use of cultural and chemical controls to manage rose downy mildew in landscapes and in greenhouse and nursery production areas.

Emergence of Groundnut ringspot virus and Tomato chlorotic spot virus in Vegetables in Florida and the Southeastern United States.

Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) are two emerging tospoviruses in Florida. In a survey of the southeastern United States, GRSV and TCSV were frequently detected in solanaceous crops and weeds with tospovirus-like symptoms in south Florida, and occurred sympatrically with Tomato spotted wilt virus (TSWV) in tomato and pepper in south Florida. TSWV was the only tospovirus detected in other survey locations, with the exceptions of GRSV from tomato (Solanum lycopersicum) in South Carolina and New York, both of which are first reports. Impatiens (Impatiens walleriana) and lettuce (Lactuca sativa) were the only non-solanaceous GRSV and/or TCSV hosts identified in experimental host range studies. Little genetic diversity was observed in GRSV and TCSV sequences, likely due to the recent introductions of both viruses. All GRSV isolates characterized were reassortants with the TCSV M RNA. In laboratory transmission studies, Frankliniella schultzei was a more efficient vector of GRSV than F. occidentalis. TCSV was acquired more efficiently than GRSV by F. occidentalis but upon acquisition, transmission frequencies were similar. Further spread of GRSV and TCSV in the United States is possible and detection of mixed infections highlights the opportunity for additional reassortment of tospovirus genomic RNAs.