First Detection of Pseudocerradoa paullula Causing Aroid Leaf Rust on Swiss Cheese Plant (Monstera deliciosa) in Virginia.

Monstera deliciosa Liebm. (Araceae) is a monocotyledonous plant that is native to tropical forests of southern Mexico to Panama. It is widely grown as an ornamental in the United States because of its easy maintenance and attractive, fenestrate leaves. On May 10th, 2024, at a nursery and garden center in Henrico County, Virginia, four M. deliciosa plants in 3.8 L containers were observed with necrotic spots surrounded by a yellow halo on the leaves (Fig. 1A). Uredinia were present in the center of the lesions with dense, reddish-brown sporulation mostly on the abaxial surface of the leaves (Fig. 1B). Urediniospores with pedicels were golden brown in color, globose, echinulate, with two opposite germ pores, averaging (28) 25.2 x 25 (23) µm (n = 40) in size and a wall thickness of 1.5 to 2 µm (n = 40) (Fig. 1F - K). Telia were not present. The host, symptoms, and urediniospore size was comparable to reports of Pseudocerradoa paullula (Syd. & P. Syd.) M. Ebinghaus & Dianese from South Carolina (22.9 to 27.9 µm), Florida (24 to 31 µm), and Japan (24.8 to 29.3 µm) (Ebinghaus et al. 2022; Sakamoto et al. 2023; Urbina et al. 2023; Yang et al. 2023). Urediniospores from the infected plants were collected with a sterile needle and DNA was extracted using a Qiagen DNeasy PowerLyzer Microbial Kit (Germantown, MD) according to the manufacturer's instructions. PCR and sequencing of the small ribosomal subunit (SSU) and large ribosomal subunit (LSU) gene regions was performed with primer sets NS1/Rust18SR and LRust1R/LR3 (Beenken et al. 2012; Vilgalys and Hester 1990). The resulting 1,630bp and 638 bp sequence fragments of the SSU and LSU loci from strain GS24-AE50 were deposited into the NCBI Genbank database under accessions PQ059898 and PQ059897, respectively. A pairwise alignment of the SSU gene shared 1,363/1,366 (99%) nucleotides with the P. paullula voucher (ON887197) from Florida. A Genbank nBLAST analysis of the LSU gene shared 636/638 (99%), 636/638 (99%), and 592/600 (99%) nucleotides with vouchers from M. deliciosa from South Carolina (OQ746460), Florida (ON887197) and Japan (OK509070) (Sakamoto et al. 20222; Urbina et al. 2023; Yang et al. 2023). Koch's postulates were fulfilled by spraying four, healthy, non-wounded M. deliciosa plants to run-off with a urediniospore suspension (1 x 106 spores/ml distilled water, 20 ml per plant) that was collected from the original infected plants. An additional four, healthy control plants were sprayed with distilled water only. After 6 weeks in a greenhouse at 22 ± 2°C with ≥85% relative humidity under an 8-h photoperiod, uredinia in the center of lesions identical to those on the original symptomatic plants developed on 12 out of 20 leaves from the inoculated plants, while all the leaves from the control plants remained asymptomatic (Fig.1C - E). Urediniospores collected from the inoculated plants were morphologically identical to the urediniospores from the original infected plants with 100% LSU sequence homology to accession PQ059897. Globally, P. paullula has been reported from Australia, China, Japan, Malaysia, the Philippines, and the United States, where the pathogen was detected at the port of Los Angeles in 2014, Florida in 2019, and South Carolina in 2023 (Sakamoto et al. 2023; Shaw et al. 1991; Urbina et al. 2023; Yang et al. 2023). Although the pathogen is not known to be established in Virginia, the recent surge of reports suggests that the pathogen's distribution is expanding. The impact of aroid leaf rust on M. deliciosa production is unclear, but it has the potential to reduce the aesthetic and commercial value of plants under favorable conditions.

First record of Diplodia gallae causing branch cankers on declining scarlet oak (Quercus coccinea) in Virginia.

Members of Botryosphaeria s.l. have an extensive history as cankering pathogens of stressed and declining oak trees in the eastern United States (Ferreira et al. 2021). The host range, distribution, and virulence among two closely related species, Diplodia corticola and D. gallae, remains unclear (Brazee et al. 2023). On 15 August 2023, a survey was conducted at a declining natural hardwood site in Shenandoah County, Virginia (GPS coordinates 38.922089, -78.606125). One mature Quercus coccinea tree that displayed scorched leaf margins and branch dieback was felled and a cankered branch from the crown was sampled (Fig. 1A and B). A 4-mm piece of necrotic tissue was selected from the margin of the canker, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, four colonies were transferred to full-strength PDA medium and incubated in the dark at 22 ± 2°C. After 10 days, all four colonies displayed thick, gray, floccose mycelium and pigmented hyphae (Fig. 1C). Mycelia was harvested from 10-day-old colonies with a sterile pin and DNA was extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A fragment of the internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY).&xa0; The raw nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). All four colonies had identical ITS sequences. A 523 and 276-bp fragment of the ITS and tef1 loci, respectively, from isolate R1.2 was deposited into the GenBank database (accessions OR934498 and OR961039). A dataset of 43 strains consisting of 38,658 characters was aligned using MAFFT v7.49 (Katoh et al. 2013), and a concatenated ITS + tef1 maximum likelihood phylogenetic tree (1000 bootstraps) was built with PhyML 3.0 (Guindon et al. 2010) using the GTR substitution model. Isolate R1.2 was grouped with isolates of D. gallae although the species failed to form a well-supported clade (BS = 67) due to intraspecific variation (Fig. 1D). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. coccinea trees (average stem caliper 5.3 cm) with isolate R1.2, with five plants as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 4°C. The same procedure was performed on the control plants using sterile PDA plugs. After five weeks the bark was removed, and all five stems treated with R1.2 had necrotic lesions with a mean linear growth ([length+width]/2) of 9.2 ± 2.72 mm from the edge of the wound, which was significantly larger (P = 0.003) than the controls (1 ± 0.66 mm; Fig. 1E - L). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. gallae based on morphology and 100% ITS sequence homology to isolate R1.2. D. gallae was not recovered from the control plants. In the United States, D. gallae has been isolated from Q. rubra and Q. velutina twig cankers in Maine, Massachusetts, New Hampshire, New York, and Vermont (Brazee et al. 2023). This is the first report of the species in Virginia causing branch cankers on Q. coccinea.

First report of Diplodia quercivora causing branch cankers on declining chestnut oak (Quercus montana) in Virginia.

Since the beginning of the twentieth century oak decline has been documented in central and eastern hardwood forests of the United States as a stress-mediated disease (Oak et al. 2016). Opportunistic canker pathogens, including Diplodia corticola, D. quercivora, D. sapinea, and Botryosphaeria dothidea have been associated with crown dieback of declining oak trees in several mid-Atlantic states (Ferreira et al. 2021). On 02 August 2022, a survey was conducted at two natural hardwood sites in Fredrick and Shenandoah Counties, Virginia that exhibited symptoms of decline (Fig. 1A). At both sites, mature Quercus montana trees were observed with bole and branch cankers, bleeding and sooty lesions, and discolored sapwood. Pycnidia were present on the margin of seven branch cankers from three trees that were felled, with hyaline, elliptical to oblong conidia 19.0 - 26.8 × 8.5 - 11.2 µm (n = 40) in size (Fig. 1C and D). Six cultures were derived from single spores that were placed on PDA medium and incubated for 10 days in the dark at 22 ± 2°C. Additionally, a 4-mm piece of necrotic tissue was selected from the margin of each of the seven cankers, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, seven colonies from each canker assayed were transferred to full-strength PDA plates and incubated for 10 days in the dark at 22 ± 2°C. Colonies derived from spores and the necrotic wood were morphologically identical, with white, aerial, floccose mycelium that turned dark gray to olivaceous after five days (Fig. 1B). DNA was individually extracted from four, 10-day-old cultures (two from spores and two from wood). Mycelia was harvested with a sterile pin and extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A segment of the internal transcribed spacer (ITS), large subunit rRNA (LSU), and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990), LR5/LROR (Vilgalys and Hester 1990), and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY). The nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). The resulting ITS sequences from the four isolates were identical. A 544-bp, 1131-bp, and 273-bp segment of the ITS, LSU, and tef1 loci from isolate GS22-DSB-17 was deposited into the GenBank database (accessions OQ597712, OQ597714, and OR754429, respectively). A Genbank BLAST analysis revealed that the ITS and tef1 fragments shared 510/516 (99%) and 271/273 (99%) nucleotides with the D. quercivora ex-type BL8 (JX894205/JX894229). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. montana trees (average stem caliper 6.5 cm) with D. qercivora isolate GS22-DSB-17, while five plants were used as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 6°C. The same procedure was performed on the control plants using sterile PDA plugs. After six weeks the bark was carefully removed, and all five stems treated with D. quercivora had necrotic lesions with a mean canker linear growth ([length+width]/2) of 15.6 mm from the edge of the wound, which was significantly larger (P = 0.001) than the controls (2.3 mm; Fig. 1E-M). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. quercivora based on morphology and 100% ITS sequence homology to isolate GS22-DSB-17. D. quercivora was not recovered from the control plants. In the United States, D. quercivora has been isolated from declining white oak trees in Maryland, Massachusetts, West Virginia, and Florida (Dreaden et al. 2014; Ferreira et al. 2021; Haines et al. 2019). More surveys are needed to understand the host range and distribution of D. quercivora in the United States, as well as the environmental and site factors that impact oak health and predispose trees to infection from opportunistic cankering pathogens.

Phytophthora spp. Associated with Appalachian Oak Forests and Waterways in Pennsylvania, with P. abietivora as a Pathogen of Five Native Woody Plant Species.

To document the distribution of potentially harmful Phytophthora spp. within Pennsylvania, the Pennsylvania Department of Agriculture collected 89 plant, 137 soil, and 48 water samples from 64 forested sites during 2018 to 2020. In total, 231 Phytophthora strains were isolated using baiting assays and identified based on morphological characteristics and sequences of nuclear and mitochondrial loci. Twenty-one Phytophthora spp. in nine clades and one unidentified species were present. Phytophthora abietivora, a recently described clade 7a species, was recovered from diseased tissue of 10 native broadleaved plants and twice from soil from 12 locations. P. abietivora is most likely endemic to Pennsylvania based on pathogenicity tests on six native plant species, intraspecific genetic diversity, wide distribution, and recoveries from Abies Mill. and Tsuga Carrière plantations dating back to 1989. Cardinal temperatures and morphological traits are provided for this species. Other taxa, in decreasing order of frequency, include P. chlamydospora, P. plurivora, P. pini, P. cinnamomi, P. xcambivora, P. irrigata, P. gonapodyides, P. cactorum, P. pseudosyringae, P. hydropathica, P. stricta, P. xstagnum, P. caryae, P. intercalaris, P. 'bitahaiensis', P. heveae, P. citrophthora, P. macilentosa, P. cryptogea, and P. riparia. Twelve species were associated with diseased plant tissues. This survey documented 53 new plant-Phytophthora associations and expanded the known distribution of some species.

First Report of Aloe vera Rust Caused by Uromyces aloes in an Ornamental Nursery in the United States.

Aloe vera (L.) Burm. f. is a tropical evergreen perennial in the family Liliaceae. Native to the Arabian Peninsula, it is sold in Pennsylvania as an ornamental and for its medical and topical purposes due to its high levels of amino acids, anthraquinones, saponins, and vitamins A, B, C, E (Sahu et al. 2013). In February 2020, at an ornamental plant nursery in Lancaster County, Pennsylvania, 5 out of 15 mature A. vera plants in 15 cm pots showed symptoms and signs of rust on the leaves, exhibiting dark-brown erumpent pycnial spots with a chlorotic band surrounding the infected tissue that turned necrotic after three days of incubation at 20°C. Only the telial stage was present. Sori (n=25) were rounded, concentrically arranged, 0.2-3.7 mm, and covered by a brown epidermis. Teliospores (n=40) were amphigenous, orange-brown, globose to ellipsoidal, measuring (29.2) 30.4-36.1 (39.5) × (27.4) 27.6-30.1 (30.5) µm, with a wall thickness of 4-5 µm, and a persistent hyaline pedicel ranging from 5 to 57.1 µm in length and 5.2 to 9.3 µm in width. These measurements were comparable to the descriptions of Uromyces aloes previously reported from India (teliospore size 25-42.5 x 20-30 µm, wall thickness 3-5 µm, and pedicel size 25-95 x 5-6.25 µm), and South Africa (teliospore size 30-44 x 24-32 µm, wall thickness 4-6 µm, and pedicel size 6-20 µm) (Maier et al. 2007; Soni et al. 2011). Based on these morphological traits and the plant host, the causal agent was identified as Uromyces aloes (Cooke) Magnus (Pucciniaceae, Uredinales). The sample was also independently identified as U. aloes by the USDA APHIS PPQ Beltsville lab (Interception # APEMD200552555001) based on morphological characteristics. Teliospores were harvested with a sterile pin, transferred to a 1.5 ml tube with DNA extraction buffer (100 mM Tris-HCL, 10 mM EDTA, 1 M KCl, pH 8) and macerated using a plastic mini-pestle. The DNA was precipitated using isopropanol, washed with 70% ethanol, and reconstituted in 50 µl of PCR-grade water. The segment of the internal transcribed spacer region (ITS) was amplified using ITS4/ITS5 primers (White et al. 1990). The nuclear ribosomal small subunit (18S) was amplified with rust specific primers Rust18S-R (Aime 2006) and NS1 (White et al. 1990). The nuclear ribosomal large subunit (28S) was amplified with primers LR0R and LR7 (Vilgalys et al. 1990). Amplified PCR products were cleaned using ExoSap (Affymetrix, Santa Clara, CA) or QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and sequenced at Penn State Genomics Core Facility. The nucleotide sequences were trimmed, analyzed, and aligned using Geneious 11.1.5 software (Biomatters, Auckland, NZ). The resulting 692-bp segment of the ITS, 1,633-bp segment of the 18S, and the 1,324-bp segment of the 28S regions were deposited in the GenBank database under accession numbers MT136509, MZ146345, and MZ146342, respectively. Based on GenBank BLAST analysis, a 529-bp fragment of our 28S product was found to share 98.87% (523/529) identity with U. aloes isolate WM3290 (DQ917740) from South Africa, with three nucleotide differences and three gaps between the two strains. Comparisons among ITS and 18S sequences could not be made because no ITS or 18S sequence data from U. aloes has previously been deposited in GenBank. To our knowledge, this is the first report of U. aloes from A. vera in the United States. Infected plants were confined inside a greenhouse and have been destroyed. Since the plants were purchased from either Ontario, Canada or Florida, the extent of infection in the United States is unknown.

Phytophthora Diversity in Pennsylvania Nurseries and Greenhouses Inferred from Clinical Samples Collected over Four Decades.

The increasing movement of exotic pathogens calls for systematic surveillance so that newly introduced pathogens can be recognized and dealt with early. A resource crucial for recognizing such pathogens is knowledge about the spatial and temporal diversity of endemic pathogens. Here, we report an effort to build this resource for Pennsylvania (PA) by characterizing the identity and distribution of Phytophthora species isolated from diverse plant species in PA nurseries and greenhouses. We identified 1137 Phytophthora isolates cultured from clinical samples of >150 plant species submitted to the PA Department of Agriculture for diagnosis from 1975 to 2019 using sequences of one or more loci and morphological characteristics. The three most commonly received plants were Abies, Rhododendron, and Pseudotsuga. Thirty-six Phytophthora species identified represent all clades, except 3 and 10, and included a distinct subgroup of a known species and a prospective new species. Prominent pathogenic species such as P. cactorum, P. cinnamomi, P. nicotianae, P. drechsleri, P. pini, P. plurivora, and P. sp. kelmania have been found consistently since 1975. One isolate cultured from Juniperus horizontalis roots did not correspond to any known species, and several other isolates also show considerable genetic variation from any authentic species or isolate. Some species were isolated from never-before-documented plants, suggesting that their host range is larger than previously thought. This survey only provides a coarse picture of historical patterns of Phytophthora encounters in PA nurseries and greenhouses because the isolation of Phytophthora was not designed for a systematic survey. However, its extensive temporal and plant coverage offers a unique insight into the association of Phytophthora with diverse plants in nurseries and greenhouses.