Live tracking of a plant pathogen outbreak reveals rapid and successive, multidecade plasmid reduction.

Quickly understanding the genomic changes that lead to pathogen emergence is necessary to launch mitigation efforts and reduce harm. In this study, we tracked in real time a 2022 bacterial plant disease outbreak in U.S. geraniums (Pelargonium × hortorum) caused by Xhp2022, a novel lineage of Xanthomonas hortorum. Genomes from 31 Xhp2022 isolates from seven states showed limited chromosomal variation and all contained a single plasmid (p93). Time tree and single nucleotide polymorphism whole-genome analysis estimated that Xhp2022 emerged within the last decade. The phylogenomic analysis determined that p93 resulted from the cointegration of three plasmids (p31, p45, and p66) found sporadically across isolates from previous outbreaks. Although p93 had a 49 kb nucleotide reduction, it retained putative fitness genes, which became predominant in the 2022 outbreak. Overall, we demonstrated, through rapid whole-genome sequencing and analysis, a recent, traceable event of genome reduction for niche adaptation typically observed over millennia in obligate and fastidious pathogens.IMPORTANCEThe geranium industry, valued at $4 million annually, faces an ongoing Xanthomonas hortorum pv. pelargonii (Xhp) pathogen outbreak. To track and describe the outbreak, we compared the genome structure across historical and globally distributed isolates. Our research revealed Xhp population has not had chromosome rearrangements since 1974 and has three distinct plasmids. In 2012, we found all three plasmids in individual Xhp isolates. However, in 2022, the three plasmids co-integrated into one plasmid named p93. p93 retained putative fitness genes but lost extraneous genomic material. Our findings show that the 2022 strain group of the bacterial plant pathogen Xanthomonas hortorum underwent a plasmid reduction. We also observed several Xanthomonas species from different years, hosts, and continents have similar plasmids to p93, possibly due to shared agricultural settings. We noticed parallels between genome efficiency and reduction that we see across millennia with obligate parasites with increased niche specificity.

Evaluation of Anaerobic Soil Disinfestation to Reduce Soilborne Diseases in Soilless and Soil-Based Substrates for Specialty Cut Flower Production.

Anaerobic soil disinfestation (ASD) is a nonchemical soil treatment where an easily decomposable carbon source is incorporated into soil, which is then irrigated to saturation and tarped to create anaerobic conditions, which prompts shifts in the soil microbiota from aerobes to anaerobes. ASD has been tested successfully for soilborne disease management in a variety of cropping systems but has not been sufficiently investigated in ornamentals. In this study, ASD was evaluated in soil-based and soilless substrates commonly used in specialty cut flower production using two model pathosystems: Rhizoctonia solani-Zinnia elegans and Phytophthora drechsleri-Gerbera jamesonii. Each substrate was mixed with pathogen-infested vermiculite and amended with either wheat bran, tomato pomace, or soybean meal as the carbon source. Amended substrates were incubated at 25°C for 4 weeks and used as growing substrates for the two crops mentioned above, which were monitored weekly for disease development for up to 5 weeks posttransplant. Additional experiments tested the effect of plant age and inoculum concentration in the substrate on ASD efficacy. Results showed that ASD has the potential to be deployed successfully for the control of Rhizoctonia stem rot in both substrates. Conversely, ASD was not effective at controlling Phytophthora crown rot on gerbera daisy in any of the experiments conducted in this study. More research is needed to understand the influence of carbon amendments, inoculum thresholds, and environmental conditions on ASD efficacy.

First Report of Pseudomonas syringae Causing Bacterial Leaf Spot on Winterberry Holly (Ilex verticillata) in Ohio.

Winterberries (Ilex verticillata and hybrids) are deciduous species of holly whose branches bearing colorful fruit are cut in late Fall to be used for seasonal decorations. The annual wholesale value of the woody cuts is $1.5 million nationally (NASS, 2019). In June 2021, approximately 80% of the 45 Ilex verticillata 'Maryland Beauty' potted plants, which were maintained in a container yard at The Ohio State University research farm in Columbus, OH, presented leaves with irregular necrotic lesions surrounded by a chlorotic halo. No other symptoms were present on the plants. Bacterial streaming was observed from the lesions using a compound microscope and isolations were performed after surface disinfesting small sections of leaf tissue from the border of the lesions by soaking in 10% bleach for 30 sec, rinsing twice in sterile water, macerating in sterile water, and streaking the suspension on nutrient broth yeast extract agar. Creamy white, circular, smooth, and convex colonies were recovered after incubation at 28°C for 48 h. Bacterial identification of one representative isolate was initially pursued from single colonies of a purified culture using five discriminative phenotypic tests (i.e., LOPAT: "L", levan production; "O", oxidase activity; "P", pectinolytic activity; "A", arginine dehydrolase production; "T", tobacco hypersensitive reaction), which resulted in the L+ O- P- A- T+ profile consistent with the description of Pseudomonas syringae (Lelliott et al. 1996). Molecular identification was performed based on rpoD marker amplification and sequencing using primers PsrpoD FNP1/PsrpoDnprpcr1 (Parkison et al. 2011). NCBI GenBank BLASTn comparison of the rpoD sequence (GenBank Acc. No. OP221440) shared 99.12% identity to P. syringae pv. passiflorae (AB163366.1). Whole genome sequence analysis was conducted to strengthen the classification of the isolate species. To this extent, DNA was sequenced with an iSeq 100 Illumina benchtop sequencer using Illumina DNA Prep kit and iSeq 100 i1 Reagent v2 (Illumina, Inc, REF: 20060060 and 20031371). Illumina Local Run Manager software was used for base calling, demultiplexing, and trimming of the raw reads. Unicycler v0.5.0 was used for de novo assembly of the genome (Wick et al. 2017). The assembled genome size was 5.9 Mb with 959 contigs and 10× coverage (NCBI GenBank Biosample No. SAMN30281368; Acc. No. JANQCB010000000). Average nucleotide identity (ANI) analysis was performed on the server MiGA online (Rodriguez-R et al. 2018). Subgroup identification was inconclusive (p>0.05), positioning this isolate between P. syringae pv. actinidiae (96.45% ANI) and pv. viburni (96.65% ANI) (Rodriguez-R & Konstantinidis, 2016). Both these pathovars cause leaf spots on woody plants such as kiwi and viburnum (Donati et al. 2020; Garibaldi et al. 2005). To confirm pathogenicity, three separate branches on each of two I. verticillata 'Maryland Beauty' potted plants were selected, and 5-7 individual young leaves (>2 weeks from emergence) on each branch were infiltrated with a bacterial suspension (108 CFU/mL) in sterile water (SW) using a needleless syringe by delivering 30-50 µL of suspension per infiltration point. One additional branch per plant was infiltrated with SW to serve as control. Plants were covered with a plastic bag for two days post-inoculation (DPI) and maintained in the laboratory at an average of 23°C. All inoculated leaves showed necrotic lesions two DPI while control leaves remained asymptomatic. To fulfill Koch's postulates, the bacterium was re-isolated from the symptomatic leaves six DPI and confirmed to be identical to the original isolate based on rpoD gene sequencing. To the best of our knowledge, this report signifies the first instance of P. syringae causing bacterial leaf spot on winterberry worldwide. Ornamental plant sales are based primarily on visual appeal; therefore, identification and monitoring of emerging pathogens is essential to ensure the health of the industry.

First Report of Colletotrichum sansevieriae Causing Anthracnose of Snake Plant (Dracaena trifasciata) in Ohio and its Draft Genome.

Dracaena trifasciata (Prain) Mabb. is a popular houseplant in the United States. In September 2021, two diseased samples from two Ohio homeowners were received by the Ornamental Pathology Laboratory at The Ohio State University. Each sample included one or two detached leaves displaying circular gray water-soaked lesions scattered throughout the lamina and blighted areas with concentric rings bearing brown to black acervuli. Lesions covered between 25 and 50% of the leaf surface. Isolations were made by excising small portions of leaf tissue from the margin of the lesions, surface-disinfesting in 10% bleach for 45 s, rinsing in sterile water, and plating on potato dextrose agar (PDA). Plates were incubated at 23°C for one week. Two representative isolates, one per sample (FPH2021-5 and -6), were obtained by transferring hyphal tips to fresh PDA plates. Mycelia of both isolates were aerial, cottony, grayish-white, producing spores in a gelatinous orange matrix, and appeared gray to olivaceous-gray on the plate underside. Conidia produced by both isolates were cylindrical, single-celled, hyaline, measuring 12.02 to 18.11 (15.51) × 5.03 to 7.29 (6.14) µm (FPH2021-5; n=50) and 15.58 to 20.90 (18.39) × 5.63 to 8.27 (7.05) µm (FPH2021-6; n=50). Appressoria were globose to subglobose, single-celled, dark brown to sepia, measuring 6.62 to 13.98 (8.97) × 5.05 to 6.58 (6.58) µm (FPH2021-5; n=50), and 6.54 to 11.32 (8.63) × 4.54 to 8.94 (7.09) µm (FPH2021-6; n=50). Genomic DNA (gDNA) samples were extracted from both isolates and the internal transcribed spacer (ITS) region was amplified using primers ITS1F/ITS4 (Gardes and Bruns, 1993; White et al. 1990). GenBank BLAST sequence analysis resulted in 99.83% (FPH2021-5; GenBank Acc. No. OP410918.1) and 100% (FPH2021-6; OP410917.1) identity with 100% query coverage to the type strain of Colletotrichum sansevieriae Miho Nakam. & Ohzono MAFF239721 or Sa-1-2 (NR_152313.1; Nakamura et al. 2006). Whole genome sequencing was conducted for FPH2021-6 and the assembly was deposited in GenBank (JAOQIF000000000.1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin (ß-tub) regions were either extracted from the genome of FPH2021-6 (OP414603.1 and OP414601.1, respectively) or amplified from FPH2021-5 gDNA using primers GDF/GDR (OP414604.1) and Bt-2b/T1 (OP414602.1), respectively (Templeton et al. 1992; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997). A multilocus partitioned analysis (Chernomor et al. 2016) based on concatenated sequences of ITS, GAPDH, and ß-tub using ModelFinder (Kalyaanamoorthy et al. 2017) was performed to build a maximum likelihood tree (IQ-TREE v2.0.3; Nguyen et al. 2015), suggesting that these two isolates are phylogenetically closer to the type strain from Japan than to a previously reported isolate 1047 from Florida (Palmateer et al. 2012). To fulfill Koch's postulates, two parallel leaf sections from one 10-inch D. trifasciata 'Laurentii' plant maintained in a 1.3-liter container were selected. Three wounds were made in each section using a sterile syringe needle. A 10-µl drop of either a 1×106 conidia/ml suspension of isolate FPH2021-6 or sterile water was placed on each wound. The plant was covered with a plastic bag for two days post-inoculation (DPI) and maintained in a greenhouse at 25°C with a 12- h photoperiod. The experiment was conducted twice. Grayish water-soaked lesions, acervuli, and leaf blight were observed on the inoculated sections 3, 10, and 14 DPI, respectively, while no symptoms appeared on the sections treated with sterile water. C. sansevieriae was re-isolated from the lesions and confirmed to be identical to the original isolate based on ITS sequencing and morphological examinations. To the best of our knowledge, this is the first report of C. sansevieriae on D. trifasciata in Ohio and the first genome draft of an isolate from the United States. Availability of whole-genome sequence data is paramount for resolving species identification in this highly diverse fungal genus, and a powerful tool to conduct comparative genomic analyses in the future.

Confirmation of Burkholderia gladioli as the Causal Agent of Bacterial Scab on Gladiolus (Gladiolus spp.) in Ohio.

Ohio is one of the top five floriculture producers in the United States, grossing over $200 million annually (NASS 2019). Within the international floriculture trade, gladiolus cut flowers represent the fifth highest grossing crop (Ahmed et al. 2002). In September 2021, the Ornamental Crops Pathology Lab at the Ohio State University received a gladiolus (Gladiolus spp.) sample of an unknown cultivar from a home garden in Franklin Co., OH where several plants had failed to grow from planted corms or were stunted and displaying symptoms of disease. Bleached, water-soaked spots with necrotic margins along the flowering stems, stunted flowers with partial necrosis, and necrotic bracts were observed on the submitted sample. Bacterial isolations were performed by surface disinfesting small sections of bract tissue from the border of a lesion by soaking in 10% bleach for 30 sec and rinsing twice in sterile water, macerating the tissue in sterile water, and streaking the suspension on nutrient agar (NA) plates. Plates were incubated at 28°C for 48 hours and the resulting colonies were purified by re-streaking a single colony on NA twice. Bacterial colony morphology on NA presented as cream-colored and shiny with an irregular form and undulate margin. Five in vitro tests were performed using one representative isolate to identify the bacterium to the genus level: (1) confirmed levan production, (2) confirmed pectinolytic activity, (3) confirmed ability to grow at 40°C, (4) inability to grow under anaerobic conditions, and (5) a negative oxidase test (Schaad et al. 2000). All test results identified the genus as Burkholderia. To identify to species level, gyrase subunit B (gyrB) and RNA polymerase subunit D (rpoD) markers were PCR amplified and sequenced using primers UP1-E/AprU, and 70F2/70R2, respectively (Maeda et al. 2006). NCBI GenBank BLASTn comparison showed that the gyrB sequence shared 99.33% identity to the type strain of B. gladioli (CP009323.1), while the rpoD sequence showed 99.53% identity (CP009322.1). Sequences were deposited in GenBank under accession numbers ON597852 (gyrB) and ON597853 (rpoD). To confirm pathogenicity, each of two Gladiolus communis 'Mini Elvira' potted plants were inoculated with two bacterial and two control treatments (3 leaves/treatment/plant) as follows: leaf infiltration with 1 mL of either (i) a distilled water-Tween 20 (0.03% v/v) bacterial suspension (106 cfu/mL) or (ii) a sterile water-Tween 20 suspension using a needle-less syringe; foliar spray with either (iii) the bacterial suspension or (iv) water-Tween suspension until run-off. Following inoculation, plants were covered for 24 hours with a plastic bag to increase humidity and favor infection and maintained in a greenhouse at an average temperature of 23°C. After 3 days, water-soaked, necrotic lesions were observed on the inoculated plants regardless of inoculation method, while control leaves remained asymptomatic. To fulfill Koch's postulates, bacteria were re-isolated from the lesions 7 days post-inoculation and confirmed to be identical to the original isolate based on rpoD gene sequencing. Bacterial scab of gladiolus was reported in Ohio in the late 1900s as caused by Pseudomonas gladioli (syn. P. marginata; Ellett, 1989). To the best of our knowledge, this report represents the first molecular identification of the causal agent as Burkholderia gladioli. In Ohio, the pathogen has also been observed causing slippery skin on onion but not officially reported in the peer-reviewed literature. Additionally, B. gladioli has been reported in other parts of the United States on orchid, corn, and rice (Keith et al. 2005; Lu et al. 2007; Nandakumar et al. 2009). Given the significant role of gladiolus within Ohio's floricultural trade, as well as the ability of this pathogen to infect other regional crops, monitoring of bacterial scab is important for floriculture and field crop growers alike.

Whole-Genome Sequence Data for the Holotype Strain of Diaporthe ilicicola, a Fungus Associated with Latent Fruit Rot in Deciduous Holly.

Diaporthe ilicicola is a newly described fungal species that is associated with latent fruit rot in deciduous holly. This announcement provides a whole-genome assembly and annotation for this plant pathogen, which will inform research on its parasitism and identification of gene clusters involved in the production of bioactive metabolites.

Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples.

Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single- and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (quantification cycle [Cq], >35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen's capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.

First Report of Gymnosporangium yamadae Causing Japanese Apple Rust on Crabapple (Malus spp.) in Ohio.

Japanese apple rust, caused by the heteroecious and demicyclic rust fungus Gymnosporangium yamadae Miyabe ex G. Yamada, can affect juniper (Juniperus spp.), where the telial stage of this disease occurs, and apple or crabapple (Malus spp.), where the aecial stage occurs (Yun, 2010). Leaf samples displaying symptoms and signs of rust disease were collected in August 2020 from 14 different crabapple cultivars ('Amerspirzam' [American Spirit®], 'Amsalzam' [American Salute™], 'Excazam' [Excalibur™], 'Guinzam' [Guinevere®], 'Hargozam' [Harvest Gold®], 'Mary Potter', 'Orange Crush', 'Prairie Maid', 'Professor Sprenger', 'Pumpkin Pie', 'Rawhide', 'Select A' [Firebird®], 'Shotizam' [Show Time™], 'Sinai Fire') in the crabapple research plot of Secrest Arboretum (Crablandia) in Wooster, OH. Samples displayed adaxial leaf lesions with brown necrotic centers surrounded by a red-yellow coloration, corresponding on the abaxial side to lesions containing brown-orange aecia, producing aeciospores, surrounded by a dark red-orange coloration (Supplemental Figure 1). One to multiple lesions were present per symptomatic leaf. DNA was extracted from symptomatic leaf tissue containing fungal material on all 14 cultivars using the DNeasy Plant Mini Kit (QIagen) and the D1/D2 region of the 28S rDNA was amplified using primers NL1 and NL4 (O'Donnell 1993) according to Dagar et al. (2011). GenBank BLAST sequence analysis of all 14 sequences resulted in 99.83-100% sequence identity to G. yamadae with with 99% query coverage (MN605735). Sequences from all samples were deposited in GenBank under Accession Nos. MW131119.2-131125.2 and MW131127.2-131132.2. Morphological features were characterized for the three representative cultivars 'Amerspirzam' (American Spirit®), 'Orange Crush' and 'Pumpkin Pie' (Supplemental Figure 2). Aecia were hypophyllous, roestelioid, with cornute, yellow-brown, peridia with lacerate sides. Peridial cells appeared yellow and were long-linear rhomboid, verrucose with long papillae, smooth outer walls and echinulate inner walls, measuring 45 - 78 × 16 - 27 µm (average 65 × 21 µm), 51 - 82 × 16 - 30 µm (average 66 × 23 µm), and 47 - 93 × 14 - 31 µm (average 64 × 24 µm), respectively (n=50 per cultivar). Aeciospores were globose, 20 - 26 × 18 - 24 µm (average 23 µm × 20 µm), 21 - 28 µm × 19 - 24 µm (average 24 µm × 21 µm), and 21 - 27 µm × 18 - 23 µm (average 23 µm × 21 µm), respectively, with a slightly coronate surface and dark yellow walls 1.6 - 2.7 µm (average 2 µm), 1.4 - 2.4 µm (average 2 µm), and 1.3 - 2.5 µm (average 1.8 µm) thick, respectively (n=50 per cultivar). The telia, known to occur on Juniperus spp., were not observed. Specimens from these three cultivars were deposited into the U.S. National Fungus Collections (BPI 923889, 923888, 923887). Japanese apple rust has been officially reported in parts of Eastern Asia and the Eastern United States and is also known to be present in parts of Far East Russia and Ontario, Canada (Yun et al., 2009; CAB International, 2008). This report constitutes the first confirmed instance of G. yamadae causing Japanese apple rust in Ohio. Because infected trees tend to be highly symptomatic, this disease poses a significant threat to the nursery and landscape industries as it can decrease the market value of ornamental varieties and affect yield and crop quality in varieties used for fruit production.

Beneficial Bacteria Identified for the Control of Botrytis cinerea in Petunia Greenhouse Production.

Botrytis cinerea infects most major greenhouse crops worldwide. With its increasing resistance to conventional fungicides and the movement of the greenhouse industry toward more sustainable production practices, alternative methods of control are needed. The objective of this study was to evaluate a collection of 60 bacterial strains through both a dual-culture assay and greenhouse trials to identify strains with biocontrol activity against B. cinerea. For the dual-culture assay, each bacterial strain was streaked on potato dextrose agar medium with B. cinerea. The B. cinerea growth reduction and the zone of inhibition were measured. Thirty-five strains reduced the growth of B. cinerea. All strains were also tested in an initial greenhouse trial in which Petunia × hybrida 'Carpet Red Bright' was sprayed and drenched with the bacteria biweekly for 6 weeks. All open flowers were tagged, and plants were inoculated with B. cinerea (1 × 104 conidia per 1 ml). Disease severity indices calculated from the daily flower gray mold severity ratings of all tagged flowers were used to identify the seven top-performing strains. These seven strains were then evaluated in a greenhouse validation trial. The methods were similar to those of the initial greenhouse trials except that replicate numbers were increased. Three strains (Pseudomonas protegens AP54, Pseudomonas chlororaphis 14B11, and Pseudomonas fluorescens 89F1) were selected for the ability to reduce B. cinerea infection in a greenhouse production setting. These strains can be used in future studies to develop additional biocontrol products for the management of B. cinerea in floriculture crops.

Whole-Genome Sequence of the Phlox Powdery Mildew Pathogen Golovinomyces magnicellulatus Strain FPH2017-1.

Powdery mildew (PM) fungi are obligate biotrophs capable of infecting diverse plant hosts, ranging from monocotyledonous agricultural crops to dicotyledonous ornamental crops. The PM lifestyle poses significant challenges for studying these pathogens in isolation from their host. We present a draft genome of Golovinomyces magnicellulatus, a host-specific PM on Phlox species.

Development and Evaluation of Laboratory Bioassays to Study Powdery Mildew Pathogens of Phlox In Vitro.

The genus Phlox consists of approximately 65 species that include some of the most prevalent ornamental plants in the temperate zone. These popular ornamentals are extremely susceptible to powdery mildew (PM) caused by the biotrophic fungi Golovinomyces magnicellulatus and Podosphaera sp. In this study, we used Phlox paniculata and P. glaberrima to develop a set of laboratory tools to study these pathogens in vitro, including a detached leaf and a micropropagated plantlet bioassay. We assessed pathogen growth under different experimental conditions, which included the use of four different media variations (1/2 MS medium amended with benzimidazole and tetracycline), three ages of pathogen culture (14, 18, and 22 days), three phenological stages of the host tissue (1st, 3rd, and 5th node leaves), placement of inoculum on both leaf surfaces (abaxial and adaxial), and three different inoculation techniques (single spore transfer, colony tapping, colony brushing). Detached P. paniculata leaves were successfully maintained on benzimidazole-amended 1/2 MS medium for up to 3 weeks. For both pathogens, the use of 18-day-old cultures resulted in a higher number of larger, higher sporulating colonies compared with 1-4 and 22-day-old cultures. The adaxial side of 3rd node leaves supported statistically significant more fungal growth compared with the adaxial side of 1st and 5th node leaves. Both pathogens also successfully infected micropropagated plantlets of P. glaberrima. These newly developed tools should facilitate in vitro studies on PM of Phlox and possibly be applicable to other ornamental species attacked by the same fungi.

Determining the Sources of Primary and Secondary Inoculum and Seasonal Inoculum Dynamics of Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Fruit rot of deciduous holly, caused by species of the genera Alternaria, Colletotrichum, Diaporthe, and Epicoccum, is affecting plant production in Midwestern and Eastern U.S. nurseries. To determine the sources of inoculum, dormant twigs and mummified fruit were collected, and leaf spot development was monitored throughout the season from three Ohio nurseries over two consecutive years. Mummified fruit was the main source of primary inoculum for species of Alternaria and Epicoccum, whereas mummified fruit and bark were equally important for species of Colletotrichum and Diaporthe. Brown, irregular leaf spots developed in the summer, and disease incidence and severity increased along with leaf and fruit development. Coalesced leaf spots eventually resulted in early plant defoliation. When tested for their pathogenicity on fruit, leaf spot isolates were able to infect wounded mature fruit and induce rot symptoms, which indicated that leaf spots could serve as a source of secondary inoculum for fruit infections. In addition, spore traps were used to monitor seasonal inoculum abundance in the nurseries. Fruit rot pathogens were captured by the spore traps throughout the season, with peak dissemination occurring during flowering. In this study, we also attempted to understand the role of environmental factors on leaf spot development. Although leaf spot incidence and severity were negatively correlated to mean maximum, minimum and average temperature, a decrease in temperature also coincided with leaf senescence. The role of temperature on leaf spot development should be further studied to fully interpret these results.

Investigations on the Timing of Fruit Infection by Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Fruit rot of deciduous holly is an emerging fungal disease that is affecting plant production across midwestern and eastern U.S. nurseries. To determine the growth stage(s) of host susceptibility to infection by the major pathogens associated with the disease, Alternaria alternata and Diaporthe ilicicola, and minor pathogens such as Colletotrichum fioriniae and Epicoccum nigrum, we conducted two sets of experiments over two consecutive seasons. In the first case we monitored the presence of the pathogens as well as disease progression in a commercial nursery under natural conditions by collecting plant tissues from the flower bud stage until fruit maturity. The target pathogens were consistently isolated from asymptomatic samples at all stages of fruit development and from symptomatic samples at fruit maturity across the 2 years of collection. A significant increase in fungal isolation frequency, primarily species of Alternaria and Colletotrichum, was observed right after flowering, but fruit rot symptoms only developed on mature fruit. In the second case we artificially inoculated containerized plants maintained outdoor at our research farm with individual or combined pathogens at different fruit developmental stages, and we assessed disease incidence on mature fruit to determine the time of host susceptibility to infection and, indirectly, whether pathogens in the fungal complex carry out latent infections. D. ilicicola could cause latent infection on deciduous holly fruit when inoculated at the full bloom and petal fall stages, and all inoculations made on wounded mature fruit resulted in fruit rot. These findings suggest that flowering represents a critical period to manage D. ilicicola infections and that mature fruit should be protected from any injury to avoid disease. In both experiments a negative correlation between disease incidence and temperature was found; however, the decrease in temperature also coincided with fruit ripening. The effects of temperature and changes in physiological properties of the fruit during maturation on disease development should be further investigated to fully interpret these findings.

Identification and Characterization of Fungal Pathogens Causing Fruit Rot of Deciduous Holly.

Cut branches of deciduous holly (Ilex spp. L.) harboring colorful berries are traditionally used as ornaments in holiday decorations. Since 2012, a fruit rot of unspecified cause has resulted in significant yield reduction and economic losses across Midwestern and Eastern U.S. nurseries. In this study, symptomatic fruit samples collected from nine different locations over five years were analyzed, and several fungal species were isolated. A combination of morphological characterization, multilocus phylogenetic analyses, and pathogenicity assays revealed that Alternaria alternata and Diaporthe ilicicola sp. nov. were the primary pathogens associated with symptomatic fruit. Other fungi including A. arborescens, Colletotrichum fioriniae, C. nymphaeae, Epicoccum nigrum, and species in the D. eres species complex appeared to be minor pathogens in this disease complex. In detached fruit pathogenicity assays testing the role of wounding and inoculum concentration on disease development, disease incidence and severity increased when fruit was wounded and inoculated with a higher inoculum concentration. These findings indicate that management strategies that can protect fruit from injury or reduce inoculum may lower disease levels in the field. This research established the basis for further studies on this emerging disease and the design of research-based management strategies. To our knowledge, it also represents the first report of species of Alternaria, Colletotrichum, Diaporthe, and Epicoccum causing fruit rot of deciduous holly.

Botryosphaeriaceae species associated with dieback and canker disease of bay laurel in northern California with the description of Dothiorella californica sp. nov.

Members of the Botryosphaeriaceae are cosmopolitan fungi that may exist as seemingly innocuous endophytes or as destructive pathogens of numerous woody hosts, including fruit and nut crops, urban ornamental trees and shrubs, and forest trees. Surveys of bay laurel in northern California have revealed symptoms of dieback and branch canker of unknown aetiology. The goals of this study were to identify and clarify the species of Botryosphaeriaceae associated with these symptoms and to confirm their pathogenicity. To understand the role of members of the Botryosphaeriaceae in the dieback and canker disease of bay laurel, 23 isolates were isolated from symptomatic wood. Phylogenetic analyses of ITS, translation elongation factor 1-α, and beta-tubulin revealed three species: Botryosphaeria dothidea, Neofusicoccum nonquaesitum, and the newly described and typified species Dothiorella californica sp. nov. When select isolates were inoculated to 2- to 3-year-old branches of Umbellularia californica in a natural forest, both B. dothidea and N. nonquaesitum were pathogenic with N. nonquaesitum producing the largest lesions at 12- and 18-months post inoculation, respectively, while Do. californica did not cause wood lesions significantly greater than the mock-inoculated controls. This study represents the first attempt to identify and test the pathogenicity of Botryosphaeriaceae species associated with dieback and canker disease of bay laurel in a northern California forest.