First report of Anthracnose caused by Colletotrichum theobromicola on Cyclamen persicum in South Carolina, USA.

Florist's cyclamen (Cyclamen persicum) is an herbaceous perennial native to the Mediterranean region and has become an increasingly popular plant around the world. Leaves of these plants are cordate-shaped with varying green and silver patterns. Flowers vary in color from white through different shades of pink, lavender, and red. In September 2022, symptoms of anthracnose including leaf spots and chlorosis, wilting, dieback, and crown and bulb rot were observed on 20 to 30% of approximately 1,000 cyclamen plants in an ornamental production nursery in Sumter County, SC. Tissue samples surrounding the necrotic crowns were excised and sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto acidified potato dextrose agar (APDA), and incubated at 25°C with 24-h photoperiod. A total of five Colletotrichum isolates, 22-0729-A, 22-0729-B, 22-0729-C, 22-0729-D, and 22-0729-E were obtained by transferring hyphal tips to new plates. The morphology of these five isolates was identical, observed as gray and black with aerial gray-white mycelia and orange-colored spore masses. Conidia (n=50) measured 19.4 ± 5.1 mm (11.7 to 27.1 mm) in length and 5.1 ± 0.8 mm (3.7 to 7.9 mm) in width. Conidia were tapered with rounded ends. Setae and irregular appressoria were infrequently observed in aged cultures (> 60-day-old). These morphological features resembled those of members of the Colletotrichum gloeosporioides species complex (Rojas et al. 2010; Weir et al. 2012). Sequence of the internal transcript spacer (ITS) region of a representative isolate 22-0729-E (GenBank accession No. OQ413075) is 99.8% (532 / 533 nt) and 100% (533 / 533 nt) identical to those of the ex-neotype of Co. theobromicola CBS124945 (JX010294) and the ex-epitype of Co. fragariae (= Co. theobromicola) CBS 142.31 (JX010286), respectively. Its glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequence is 99.6% (272 / 273 nt) identical to those of CBS124945 (JX010006) and CBS 142.31 (JX010024). Its actin (ACT) gene sequence shares 99.7% (281 / 282 nt) and 100% (282 / 282 nt) identities with those of CBS124945 (JX009444) and CBS 142.31 (JX009516), respectively. Lastly, its beta-tubulin 2 (TUB2) gene sequence is 99.6% (704 / 707 nt) and 100% (707 / 707 nt) identical to those of CBS124945 (JX010447) and CBS 142.31 (JX010373), respectively. The causal agent causing anthracnose on cyclamen in SC was identified as Co. theobromicola. To confirm the pathogenicity, cyclamen 'Verano Red' plants grown in 2.5-inch pots were used in two pathogenicity assays using different inoculation methods. In the first assay, three plants were inoculated by spraying a conidial suspension (1 × 106 conidia per ml; 30 ml per plant) of isolate 22-0729-E onto the foliage. Three non-inoculated control plants were sprayed with distilled water. All six plants were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 7 days to maintain humidity. Early symptoms including small spots, marginal necrosis, and chlorosis were observed on leaves and flowers 8 days after inoculation (DAI) and the entire aboveground tissues of inoculated plants were blighted 13 to 21 DAI. Non-inoculated plants remained asymptomatic. In the second assay, sterile toothpicks were used to slightly wound the crown and bulb surface of three plants and secure a mycelial APDA plug of isolate 22-0729-E (5×5 mm2) onto each wound (three wounds per plant). Three control plants were wounded in the same manner, while sterile APDA plugs were used in place of mycelial plugs. All six plants were maintained in the same manner as in the first assay. Apparent leaf yellowing and wilting symptoms appeared as early as 13 DAI. On 21 to 28 DAI, severe crown rot on inoculated plants caused the entire foliage to collapse. At least one third of the inner crown and bulb tissues of each inoculated plant were rotten, while those of non-inoculated plants appeared healthy. Each assay was repeated once. Colletotrichum isolates resembling morphological characters of 22-0729-E were recovered from leaves and inner crown tissues of all inoculated plants in both assays, respectively, but not from non-inoculated control plants. Anthracnose diseases on Cyclamen persicum caused by Co. theobromicola (syn. Co. fragariae) have been reported in NC, USA (Lui et al. 2011) and Israel (Sharma et al. 2016). This is the first report of anthracnose on cyclamen in SC, USA. Colletotrichum gloeosporioides (teleomorph Glomerella cingulate) species complex on cyclamen has also been reported in Argentina (Wright et al. 2006), South Africa, and several other U.S. states (Farr and Rossman 2022). However, it remains unknown whether these previous reports in fact attributed to Co. theobromicola due to lack of molecular identification (Weir et al. 2012). Colletotrichum theobromicola can cause diseases on at least 30 other agricultural and horticultural crops such as strawberry, cacao, and boxwood (Farr and Rossman 2022). It may pose a threat to cyclamen in greenhouse and nursery productions. Therefore, management strategies are warranted in the future.

First report of Pseudocerradoa paullula causing aroid leaf rust on Swiss cheese plant Monstera deliciosa in South Carolina, USA.

In February 2023, two Monstera deliciosa Liebm. (Araceae) plants with typical symptoms of leaf rust disease were detected at a grocery store in Oconee Co., South Carolina. Symptoms included chlorotic leaf spots and abundant brownish uredinia, mainly on the adaxial surface of more than 50% of leaves. The same disease was detected on 11 out of 481 M. deliciosa plants in a greenhouse at a plant nursery located in York Co., South Carolina, in March 2023. The first plant sample detected in February was used for morphological characterization, molecular identification, and pathogenicity confirmation of the rust fungus. Urediniospores were densely aggregated, globose, golden to golden brown in color, and measured 22.9 to 27.9 µm (aver. 26.0 ± 1.1 µm; n=50) in diameter with wall thickness at 1.3 to 2.6 µm (aver. 1.8 ± 0.3 µm; n=50). Telia were not observed. These morphological traits aligned with those of Pseudocerradoa paullula (basionym: Puccinia paullula; Ebinghaus et al. 2022; Sakamoto et al. 2023; Sydow and Sydow 1913; Urbina et al. 2023). Genomic DNA was extracted from urediniospores collected from the naturally infected plant sample and used for PCR amplification and DNA sequencing of the large subunit (LSU) genetic marker with primers LRust1R and LR3 (Vilgalys and Hester 1990; Beenken et al. 2012). The LSU sequence of the rust fungus in South Carolina (GenBank accession: OQ746460) is 99.9% identical to that of Ps. paullula voucher BPI 893085 (763/764 nt.; KY764151), 99.4% identical to that of voucher PIGH 17154 in Florida, USA (760/765 nt.; OQ275201), and 99% identical to that of voucher TNS-F-82075 in Japan (715/722 nt.; OK509071). Based on its morphological and molecular characteristics, the causal agent was identified as Ps. paullula. This pathogen identification was also corroborated by the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Plant Pathogen Confirmatory Diagnostics Laboratory in Laurel, Maryland. To confirm the fungus's pathogenicity on M. deliciosa and M. adansonii Schott (Sakamoto et al. 2023), three plants of each Monstera species were inoculated by spraying with a suspension of urediniospores collected from the original plant sample (1 × 106 spores per ml; approx. 40 ml per plant). Three non-inoculated control plants of each host species were treated with deionized water in the same manner. Plants were placed in a plastic tray with wet paper towels to maintain moisture. The tray was placed at 22C for an 8-h photoperiod and covered for five days to facilitate infection. On 25 days after inoculation, abundant spots bearing urediniospores were produced on all leaves of inoculated M. deliciosa plants. A few uredinia were observed on two of the three inoculated M. adansonii plants. All non-inoculated control plants remained asymptomatic. Morphological features of urediniospores collected from inoculated plants matched those of Ps. paullula used as the inoculum. Aroid leaf rust on Monstera plants was officially reported in Australia, China, Japan, Malaysia, Philippines, and Florida, USA (Shaw 1991; Sakamoto et al. 2023; Urbina et al. 2023). This is the first report of Ps. paullula causing this disease on M. deliciosa in South Carolina, USA. Monstera species are popular indoor and landscape plants. Potential impact and regulatory responses regarding Ps. paullula, a newly introduced and rapidly spreading pathogen in the USA, warrant further evaluation and discussion.

First report of Sclerotinia sclerotiorum causing stem canker on industrial hemp in South Carolina, USA.

On Jun. 20th, 2022, thirty industrial hemp (Cannabis sativa L.) plants (cv. Peach Haze) were vegetatively propagated, grown in a greenhouse for 21 days, and transplanted to a field at The Hemp Mine located in Fair Play, SC. Near the time of harvest (Nov. 17th, 2022), significant mycelial growth was noticed within the floral structure on 30% of plants. Three diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Stem cankers were observed on all three plants. Sclerotia typical of Sclerotinia spp. were found inside the stems of two plants. Two pure isolates were obtained by placing a sclerotium from each plant onto an acidified potato dextrose agar (APDA) plate and transferring a hyphal tip to a new APDA plate. After a 7-day-long growth at 25°C under a 24-h photoperiod, both isolates (22-1002-A and B) produced white and sparse mycelia and dark brownish to blackish sclerotia typical of S. sclerotiorum (aver. 36.5 per 90-mm plate). Sclerotia (n=50) were spherical (46%), oval (46%), or irregular (8%) and measured 1.8 to 7.2 × 1.6 to 4.5 mm (aver. 3.6 ± 1.2 × 2.7 ± 0.6 mm). No spores were produced. Sequences of the internal transcribed spacer region including the 5.8S ribosomal RNA gene (GenBank accession no. OQ749889) and the glyceraldehyde 3-phosphate dehydrogenases (G3PDH) gene (OQ790148) of 22-1002-A are 99.8% and 100% identical to those of a S. sclerotiorum isolate LAS01 on industrial hemp (MW079844 and MW082601; Garfinkel 2021). The G3PDH sequence of 22-1002-A is also 100% identical to that of ATCC 18683 (JQ036048), an authenticated S. sclerotiorum strain used for whole genome sequencing (Derbyshire et al. 2017). Ten healthy 'Peach Haze' plants (approx. 10 to 15" tall) grown in 6" pots were used in a pathogenicity test. The epidermis layer of each main stem was slightly wounded (2 × 2 mm2, 1 mm deep) using a sterile dissecting blade. A 5 × 5 mm2 mycelial plug of 22-1002-A was placed onto the wound of each of five plants, while APDA plugs were used for five control plants. Parafilm was used to secure mycelial and sterile agar plugs. All plants were maintained in an indoor controlled environment (25°C, >60% humidity, 24-h photoperiod). Stem cankers were visible on all inoculated plants 5 days after inoculation (DAI). Four of the five inoculated plants had noticeable yellowing and wilting on the foliage 9 DAI, while control plants remained asymptomatic. Elongated and tan-colored cankers (44.3 to 86.2 mm long, aver. 63.1 ± 18.3 mm) were developed at the wounded sites of inoculated plants. Wounded sites of control plants remained green in color and only slightly expanded in length (aver. 3.6 ± 0.8 mm). Tissue was excised from the canker margin of each inoculated plant and the wounded site of each control plant, surface sterilized with 10% bleach for 1 min, rinsed in sterile water, placed onto APDA, and incubated at 25°C. Sclerotia-producing colonies typical of S. sclerotiorum were recovered from all inoculated plants after 6 days, but not from any control plants. Sclerotinia sclerotiorum has a host range of more than 400 plant species (Boland and Hall 1994). This fungus causing stem canker on industrial hemp were reported from MT (Shaw 1973) and OR (Garfinkel 2021) in the USA and Canada (Bains et al. 2000). This is the first report of this disease in SC. Industrial hemp is an emerging crop in SC. The detection of this disease helps SC growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

First report of Alternaria cinerariae causing leaf blight on Farfugium japonicum in South Carolina, USA.

Farfugium japonicum, commonly known as leopard plant, is a popular perennial used in landscapes in the Southeastern U.S. In March 2022, leaf blight was observed on 20 leopard plants at a landscape site in Georgetown Co., SC. Almost all leaves were infected. Symptoms included purple to brown necrotic leaf spots and blighted petioles. Large spots had concentric circles and coalesced causing entire leaves to blight. Leaf pieces surrounding necrotic spots were excised, sterilized in 10% bleach for 1 min, rinsed in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C. A total of three Alternaria isolates, 22-094-A, 22-094-B, and 22-094-C were obtained by transferring hyphal tips to new plates. All isolates had identical morphological traits. Colonies on PDA were blackish at the center and brownish at the edge. Conidia were produced using a technique described by Shahin and Shepard (1979). Conidiophores were mostly short and unbranched. They were characterized by solitary conidia or short chains of two to three conidia. Conidia (n=30) were obpyriform to obclavate and averaged 88.5 ± 26.1 µm in body length, 118.4 ± 36.3 µm in total length, and 23.9 ± 5.9 µm in width. They had 3 to 7 transverse septa and 0 to 4 longitudinal septa. Beaks were broadly tapered. Sequence of the internal transcript spacer (ITS) region of isolate 22-094-A (GenBank Accession No. OP481973) had 100% homology to that of CBS 116495 (KC584190), a representative strain of A. cinerariae (Woudenberg et al. 2013). Based on the morphological and sequence characters, the casual fungus was identified as A. cinerariae. Pathogenicity confirmation was done in two separate assays. In a detached-leaf assay, mature leaves were collected from 5-year-old F. japonicum 'Gigantea' plants. Five leaves (abaxial surface) were sprayed with a mixture of conidial suspensions of the three isolates at 300 conidia per mL and 1.5 mL per leaf, while sterile water was used for a non-inoculated control leaf. Leaves were placed in a plastic tray with wet paper towels. The tray was placed at 22°C for an 8-h photoperiod and covered for 3 days to maintain moisture. Small purple to brown spots were visible on inoculated leaves 2 days after inoculation (DAI). More than 90% of inoculated leaf areas were blighted 10 DAI, whereas the control leaf remained asymptomatic. In a whole-plant assay, three F. japonicum 'Argenteo Marginata' plants grown in 10-inch pots were placed in a plastic tray and sprayed with a conidial suspension of 22-094-A onto both abaxial and adaxial surfaces at 300 conidia per mL and 40 mL per plant. The tray was maintained as described above. Sterile water was used for a non-inoculated control plant. Small leaf spots appeared on the inoculated plants 2 DAI. Large necrotic areas developed on leaves and girdled petioles causing aboveground tissues to collapse 4 DAI. All inoculated leaves were blighted 7 to 10 DAI, while the non-inoculated control plant remained healthy. Each assay was repeated once. Alternaria cinerariae, identified by distinct morphology traits (Nishikawa and Nakashima 2015), was consistently re-isolated from inoculated leaves in both assays. Leaf spot on F. japonicum caused by A. cinerariae has been reported in CA, USA (Woudenberg et al. 2013) and Japan (Sakoda et al. 2010). This is the first report in SC, USA. This fungus also infects at least 25 other hosts (Farr and Rossman 2022). This disease may pose a threat to leopard plants in nurseries and landscapes under conducive conditions. Disease management strategies are warranted.