Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Identification of Metabolites in Winterberry Fruit Putatively Associated with Natural Disease Resistance to Diaporthe ilicicola.

Winterberry holly (Ilex verticillata) is an ornamental plant popularly used in landscape design and sold as cut branches for fall and winter seasonal decoration. Latent fruit rot of winterberry is an emerging disease caused by the fungus Diaporthe ilicicola, which can result in up to 100% crop loss. Diaporthe ilicicola infects open flowers in spring, but symptom onset does not occur until the end of the growing season when the fruit is fully mature. This study was conducted to identify compounds displaying significant variation in abundance during fruit maturation and that may be putatively associated with natural disease resistance observed when the fruit is immature. Winterberry 'Sparkleberry' fruits collected at four timepoints during the 2018 and 2019 seasons were extracted in methanol and analyzed using high resolution ultra-high performance liquid chromatography-tandem mass spectrometry. The results showed a distinct separation of metabolic profiles based on fruit phenological stage. The top 100 features that were differentially expressed between immature and mature fruit were selected from both electrospray ionization (ESI) (-) and ESI (+) datasets for annotation. Eleven compounds shown to decrease throughout the season included cinnamic acids, a triterpenoid, terpene lactones, stilbene glycosides, a cyanidin glycoside, and a furopyran. Nine compounds shown to accumulate throughout the season included chlorogenic acid derivatives, hydrolysable tannins, flavonoid glycosides, and a triterpene saponin. Future research will further confirm the exact identity of the compounds of interest and determine whether they are biologically active toward D. ilicicola or I. verticillata. The results could inform breeding programs, chemical management programs, and novel antifungal compound development pipelines.

Understanding Environmental and Physiological Factors Affecting the Biology of Diaporthe ilicicola, the Fungus Causing Latent Fruit Rot in Winterberry.

Fruit rot in winterberry is associated with a complex of fungal pathogens. Among them, Diaporthe ilicicola plays a unique role by infecting flowers at bloom, resulting in symptom development in mature fruit. This research aimed to identify at what stage of maturation Ilex fruit can develop disease symptoms and correlate changes in fruit physiology (sugar and phenolic content) and environment (temperature and light intensity) with disease incidence. Correlation data informed in vitro studies testing the ability of putative factors to alter growth of D. ilicicola and select opportunistic fungi within the fruit rot complex: Alternaria alternata, Colletotrichum fioriniae, and Epicoccum nigrum. Results indicated that Ilex fruit do not develop symptoms until 81 to 108 days after inoculation. Temperature and fruit phenolic content were negatively correlated with disease incidence, while fruit sugar concentration and light intensity were positively correlated. In vitro assays revealed that sugar concentration had no effect on the growth of D. ilicicola, but increased light intensity increased hyphal growth and pycnidium formation. Additionally, phenolics extracted from fruit inhibited spore germination in A. alternata, induced secondary conidiation in C. fioriniae, and late season phenolic extracts increased hyphal melanization and pycnidial formation in D. ilicicola. Finally, drops in field temperatures, when replicated in vitro, resulted in a decrease in hyphal growth and spore germination for all fungi. These results suggest that changes in Ilex fruit phenolics during maturation and the increased exposure to light following defoliation may play a role in symptom development by altering D. ilicicola growth within the fruit.

Bedding Plant Production and the Challenge of Fungal Diseases.

Bedding plants are a major group of ornamentals produced in greenhouses or nurseries worldwide and planted outdoors. Their economic importance has increased continuously in the last four decades in both the United States and the European Union. These plants are subject to a broad number of diseases that can negatively impact their production and cultivation. The initial steps of production strongly influence the health status of these plants and, consequently, their aesthetic appeal, which is a strong requisite for consumers. Seeds, cuttings, and other forms of propagative material, along with production systems and growing media, can influence the phytosanitary status of the final product. In this article, case studies of soilborne and foliar diseases are presented together with preventive measures to achieve innovative disease management strategies. Quarantine restrictions and eradication measures are also discussed, in consideration of the high likelihood for ornamental plants to be long-distance vectors of new pathogens and pests.

Genome Resource: Ralstonia solanacearum Phylotype II Sequevar 1 (Race 3 Biovar 2) Strain UW848 From the 2020 U.S. Geranium Introduction.

Ralstonia solanacearum phylotype II sequevar 1 (RsII-1, formerly race 3 biovar 2) causes tomato bacterial wilt, potato brown rot, and Southern wilt of geranium. Strains in RsII-1 cause wilting in potato and tomato at cooler temperatures than tropical lowland R. solanacearum strains. Although periodically introduced, RsII-1 has not established in the United States. This pathogen is of quarantine concern and listed as a Federal Select Agent. We report a rapidly sequenced (<2 days) draft genome of UW848, a RsII-1 isolate introduced to the United States in geranium cuttings in spring 2020. UW848 belongs to the near-clonal cluster of RsII-1 global pandemic strains.

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.

Olive Twig and Branch Dieback in California Caused by Cytospora oleicola and the Newly Described Species Cytospora olivarum sp. nov.

Field surveys conducted throughout California olive-growing regions in 2008 and 2009 resulted in a collection of 101 Cytospora-like isolates from olive twig and branch dieback symptoms. Cytospora isolates were isolated from multiple cvs. in different olive orchards in Fresno, Madera, Merced, Napa, Riverside, Santa Barbara, Sonoma, Tulare, and Ventura counties. Taxonomic studies of macro- and microscopic structures along with multigene phylogenetic analyses of the internal transcribed spacer region, including the 5.8S rDNA (ITS1-5.8S-ITS2), and fragments of the translation elongation factor 1-α, beta-tubulin, and actin genes identified two species, Cytospora oleicola and C. olivarum sp. nov. Pathogenicity studies conducted in mature olive trees cvs. Manzanillo and Sevillano showed both species to be pathogenic and able to cause vascular necrosis and cankers in olive branches. This study adds to the current knowledge on the etiology of olive twig and branch dieback and provides new important information for the development of effective control strategies against canker diseases affecting olive in California.

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.

Early Detection of Airborne Inoculum of Magnaporthe oryzae in Turfgrass Fields Using a Quantitative LAMP Assay.

Gray leaf spot (GLS) is a destructive disease of perennial ryegrass caused by a host specific pathotype of the ascomycete Magnaporthe oryzae. Early diagnosis is crucial for effective disease management and the implementation of Integrated Pest Management practices. However, a rapid protocol for the detection of low levels of airborne inoculum is still missing. We developed a pathogen-specific quantitative loop-mediated isothermal amplification (qLAMP) assay coupled with a spore trap system for rapid detection and quantification of airborne inoculum of the M. oryzae perennial ryegrass pathotype, and tested its suitability for implementation in GLS-infected turfgrass fields. In summer 2015, two perennial ryegrass plots were artificially inoculated with the pathogen, with four continuously running custom impaction spore traps placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay, while plots were monitored for symptom development. Results confirmed that the qLAMP assay-trap system was able to detect as few as 10 conidia up to 12 days before symptoms developed in the field. LAMP technology is particularly appropriate for field implementation by nontechnical users, and has the potential to be a powerful decision support tool to guide timing of fungicide applications for GLS management.

The genus Cryptosphaeria in the western United States: taxonomy, multilocus phylogeny and a new species, C. multicontinentalis.

This study investigates the diversity and taxonomy of Cryptosphaeria species occurring in the western United States on the basis of morphological characters and multilocus phylogenetic analyses of the ribosomal internal transcribed spacer region, parts of a ß-tubulin gene, the DNA-dependent RNA polymerase II second-largest subunit gene and the nuclear ribosomal large subunit gene. Cryptosphaeria multicontinentalis sp. nov is described from the Sierra Nevada and central coast of California on Populus tremuloides, P. balsamifera subsp. trichocarpa and P. fremontii. Cryptosphaeria pullmanensis is reported from a wide geographic area in the western United States on the main host, P. fremontii. The pathogen C. lignyota is reported for the first time from the Sierra Nevada of California on P. tremuloides. The phylogenetic analyses showed that C. multicontinentalis is a sister species to C. lignyota. Both species were closely related to C. subcutanea and more distantly related to C. pullmanensis. Characteristics of both teleomorph and anamorph of the newly introduced species C. multicontinentalis are described and illustrated.