Acidified Potato Dextrose Agar Research Articles

Southern blight of water lily: The first host record of Agroathelia rolfsii on Nelumbo nucifera discovered in Florida, USA.

Nelumbo nucifera Gaertn. (Nelumbonaceae, Eudicots), also known as water lily or sacred lotus, is a nonnative and invasive plant commonly found in artificial ponds and natural lakes throughout Florida (UF-IFAS 2023; Wunderlin et al. 2023). In August 2020, a single sample of water lily plants showing large leaf spots were collected at a residence in Dunnellon, Marion County, Florida (80% disease prevalence with 40% leaf coverage). Symptoms and signs of the disease were necrotized adaxial leaf spots only, bordered by whitish mycelia and hyphae with clamp connections, and whitish to light brown sclerotia formed in the center (<0.7 mm diameter). Symptomatic tissue was plated on acid potato dextrose agar (APDA) amended with chloramphenicol (100 mg/L) and ampicillin (30mg/L), and incubated at 20 °C for one week. Data supporting the molecular identification of this putative pathogen were gathered by PCR amplification and Sanger sequencing of the complete internal transcribed spacer (ITS) and a fragment of the large subunit (LSU) of the rRNA gene (~1.5 kb) using primers ITS1F and LR5 (FDACS-DPI PPST 2020-105211, GenBank OR492009) (White et al. 1990). The identification of the host was confirmed by Sanger sequencing of three plant barcode fragments: ITS2 (ITS2-S2F/ITS4, OR492008), ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) (rbcLa-F/rbcLa-R, GenBank OR502388), and Maturase K (matK) (matK-KIM1R/matK-KIM3F, GenBank OR502389) (Fazekas et al. 2012). MegaBLAST queries of the ITS/LSU sequence obtained here recovered a 99.61% match to the fungal pathogen Agroathelia (=Athelia) rolfsii (Sacc.) Redhead & Mullineux. (Redhead and Mullineux 2023) (Amylocorticiaceae, Agaricomycotina) strain GP3 (GenBank JABRWF010000005) (Yan et al. 2021). MegaBLAST queries of three host plant DNA barcodes recovered matches of greater than 99.62% similarity to N. nucifera sequences. After diagnosis, symptomatic dried leaf samples were deposited at Plant Industry Herbarium Gainesville (PIHG 17807) and an axenic culture was deposited at the Agricultural Research Services Culture Collection (NRRL 66964). Koch's postulates were fulfilled by the inoculation of sclerotia (as in Terrones-Salgado et al. 2022) on adaxial leaf surface of four-week- old water lily transplants obtained from an artificial pond on campus (two plants with five leaves each). One additional transplant was not inoculated and served as a control; this plant remained asymptomatic during the experimentation period. Each transplant was kept in a 27-gallon plastic container (21W × 30L × 14H in) filled with tap water containing one tablespoon of 20-20-20 all-purpose-water-soluble plant fertilizer (VPG, TX, USA) in a plant biosafety level 2 greenhouse (23 °C, >50% relative humidity, and a 12-h/12-h photoperiod). All inoculated leaves showed necrotized areas after one week and new sclerotia were observed floating on the water surface after three weeks. Fungal pathogen was reisolated and reidentified subsequently. Agroathelia rolfsii is the causal agent of southern blight, also known as grey rot, and is reported from at least in 260 plant genera, including specialty crops such as citrus, cucumber, pepper, peanuts, pumpkin, and strawberry (Farr and Rossman 2018). Agroathelia rolfsii usually causes lower stem, crown, and root rots; consequently, leaf spots are a noteworthy presentation of symptoms for this fungus.

First report of Bretziella fagacearum infecting chestnut in Michigan.

In the summer of 2021, a 20-year-old 'Colossal' (Castanea sativa × C. crenata hybrid) tree in a commercial chestnut orchard in northwest Michigan suddenly declined. Until 2023, an additional 26 adjacent trees declined, suggesting the occurrence of root-graft transmission of the pathogen. The initial wilting of leaves progressed to complete tree death in about 10 days. Symptoms included wilting, and bronzing, followed by tanning starting at leaf apex and margins, with significant defoliation. Sometimes black-to-brown streaks of discoloration appear in the sapwood, with no signs of mycelial mat production on dead trees. Branches from symptomatic trees in two different areas of the orchard were submitted to Plant and Pest Diagnostics at Michigan State University. Bretziella fagacearum (Bretz) Z.W. de Beer, Marinc., T.A. Duong & M.J. Wingf. was detected in both samples using nested PCR (Wu et al. 2011) and qPCR (Bourgault et al. 2022). The products of the nested PCR were sequenced (GenBank accession nos. OR522695-OR522696) and BLASTn search results showed 100% identity to an ex-type strain of B. fagacearum (MH865866). Surface-sterilized discolored sapwood chips were plated on acidified potato dextrose agar (aPDA). Bretziella fagacearum was consistently recovered; colony and endoconidia morphology aligned with the description of the pathogen (De Beer et al. 2017). A pure culture (BF277) was obtained for inoculation experiments. To confirm pathogenicity, 10 'Colossal' chestnut seedlings (average stem diameter of 9 mm) were inoculated in the greenhouse with a 14-day old culture of BF277. Using a conical drill bit, two 0.4 mm diameter holes were drilled, one was 5 cm above the soil line at a 45° angle and the other was on the opposite side of the stem at least 10 cm above the soil line. A 50-µl conidial suspension (1 × 107 conidia per ml) was applied and the holes were sealed with Parafilm. Five 'Colossal' seedlings were inoculated with sterile water. Leaf epinasty with bent petioles was observed 14 days later. Leaf wilting and necrosis similar to natural infection in the orchard were observed at 24 and 34 days after inoculation, respectively. Water-inoculated control plants showed no symptoms. Bretziella fagacearum was reisolated from symptomatic plants by surface sterilizing leaf petioles with 75% ethanol (30 s), followed by 10% (v/v) bleach (1 min), and two rinses with sterile deionized water (>1 min). Petiole pieces (~1 cm) were plated on aPDA. The pathogen was reisolated from six symptomatic plants and detected using qPCR in the remaining four seedlings. Bretziella fagacearum was not detected in control plants. The identity of the recovered fungus was confirmed following the amplification of the internal transcribed spacer (ITS) from extracted genomic DNA, as described in Chahal et al. 2022. The resulting PCR product was sequenced and assembled into a consensus sequence using Geneious Prime. The consensus sequence (accession no. OR515809) revealed 100% identity to the ex-type of B. fagacearum (KU042044). This is the first record of B. fagacearum infecting chestnut trees in Michigan. Previously, B. fagacearum has been reported infecting Chinese chestnut (C. mollissima) in Missouri (Bretz and Long, 1950). Oak wilt is widely distributed in Michigan and is the predominant disease afflicting red oaks in the Midwestern U.S. Consequently, constant vigilance and monitoring are essential in chestnut orchards to promptly detect and effectively manage potential infections.

First report of Cryptostroma corticale causing sooty bark disease in California and first worldwide report of silver maple as a host.

In November 2022 and February 2023, CAL FIRE tree health experts examined four maples (Acer spp.) planted decades earlier in a residential setting in Elk Grove, Sacramento Co., California (USA). Three of the trees were silver maples (Acer saccharinum ) and one was a Norway maple (A. platanoides); both species are exotic to California. The trees were in an irreversible state of decline, with the canopy substantially thinned and browning. Extensive bark cankers elongating longitudinally along the stem were visible on all trees (Fig. S1). Cankers were filled by fungal stromata protruding through the bark and producing masses of elliptical dark brown conidia (Fig. S2), approximately 5.5 x 3.7 um in size, giving the cankers a sooty appearance. The cankered bark could be peeled off easily, revealing dark and discrete lesions in the phloem and xylem. Samples from the three trees were shipped to the U.C. Berkeley Forest Pathology and Mycology Laboratory and to the CDFA PPDC in Sacramento, CA. In the laboratories, small wood chips were taken from the margins of the lesions, surface sterilized by dipping them for 30 seconds in 70% Ethanol, rinsed for 30 seconds in sterile water and plated onto 2.5% Malt Extract Agar amended with 0.3g/L Streptomycin or onto one-half strength acidified potato dextrose agar (APDA). Two morphologically identical cultures were obtained, one (T2) from a silver maple and one (T6) from the Norway maple. Cultures were then grown in liquid 2.5% malt extract broth and, after one week, DNA was extracted using the Qiagen Plant DNeasy DNA extraction kit. The ITS sequences are diagnostic for this fungus (Li et al. 2021) and those of the two cultures (GB OR064033 and OR933565) were 100% homologous to GenBank sequences of Cryptostroma corticale ( e.g. GB OP474010-11). The RPB2 sequence of T2 ( GB OR992132) was 100% homologous to that of C. corticale (GB HG934116.1). The isolate obtained from silver maple was inoculated in four potted silver maples by removing a bark disk 50 mm in diameter with a cork borer in three spots staggered at different heights and sides on the stem, placing a colonized agar plug of C. corticale in contact with the phloem, replacing the bark disk and wrapping with parafilm. Two control trees were mock inoculated using sterile agar plugs. Trees were in 57 L pots, had an average stem caliper of 2.7 cm, an average height of 3.5 m and were kept in a lath house at average high temperatures of 18-24 degrees C. After ten weeks, average lesion length was 15.4 cm (SE= 4.6) and 4.3 cm (SE=2.3) in the fungus-inoculated and control trees, respectively. An ANOVA test, nesting lesions sizes within tree, determined lesions lengths were different between inoculated and mock trees (P= 0.04). The fungus was reisolated from all points in all inoculated trees but never from control trees. C. corticale was first described in the UK from sycamore maple (Acer pseudoplatanus) (Gregory et al. 1949) and is an emerging problem in Europe (Muller et al. 2023). In North America, it has been reported from A. negundo, A. campestre, A. macrophyllum and Cornus nuttallii (Worral 2020), and it appears to be present in the Pacific Northwest (Brooks et al. 2023, Goree 1969). Norway maple is included in the European Plant Protection Organization list of hosts for C. corticale (EPPO 2023), however our finding of C. corticale on silver maple is a first report of this host worldwide and of this pathogen in California. This report is noteworthy, given that C. corticale is also a human pathogen infecting the respiratory system (Braun et al. 2021).

Microbiological Safety and Nutritional Adequacy of Composite Porridge Flour Used in a Selected School-Feeding Program in Kamuli District, Uganda

By providing nutrient-enriched meals, the Iowa State University-Uganda Program plays a crucial role in combating hunger and improving school attendance by pupils in Kamuli district, Uganda. The lunchtime meals for pupils in primary grades 3 to 7 consist of a blend of maize grains and beans cooked together with vegetables and eggs to enhance their nutritional value. The program introduced a new composite porridge flour (44% maize, 26% millet, and 18% amaranth grain) for feeding pupils from kindergarten to primary grade 2 at break time. However, the microbiological safety of the flour for making the porridge had yet to be assessed. Additionally, though the proportions of the flour ingredients were known, its proximate composition and energy density were unknown. Hence, the porridge’s contribution to each pupils recommended dietary allowance had never been evaluated for adequacy. This study, therefore, assessed the microbiological safety and nutritional adequacy of the composite flour (n = 5 batches). Fungal contamination was determined by spread plating on acidified Potato Dextrose agar. Aflatoxin concentrations were determined by Liquid Chromatography Tandem Mass Spectrometry. Proximate composition was determined following standard methods. Gross energy was estimated using the energy conversion factors. To evaluate nutritional adequacy, the contribution of the porridge flour to pupils’ RDA was compared with the 1998 FAO/WHO RDA guidelines. The mean fungal count (4.4 ± 0.1 log cfu/g) slightly exceeded the acceptable maximum limit (4 log cfu/g), although no aflatoxins were detected, thus indicating satisfactory microbial safety. The flour predominantly contained carbohydrates (81.0% ± 0.7), moderate amounts of protein (8.2% ± 0.1), and crude fat (7.6% ± 0.5) alongside minimal levels of total ash (1.8% ± 0.1) and crude fiber (1.7% ± 0.6). Its contribution to the pupils’ RDA was very minimal owing to the low porridge intake (about 10 g dry matter per pupil per day). Therefore, there is an urgent need to enhance the nutrient profile of the composite flour by incorporating more protein and fiber rich ingredients and increasing the serving size to meet the dietary needs of the pupils. The flour could also be extruded to ensure a higher bulk density of the porridge.

First Report of Calosphaeria pulchella causing Canker and Twig Dieback of Peach (Prunus persica) in California, U.S.A.

California leads the United States in peach (Prunus persica L.) production, with approximately 505,000 tons produced in 2021 and valued at $378.3 million (California Agriculture Statistics Review, 2021-2022). During the spring and summer of 2023, twig and branch dieback were observed in three peach orchards (cvs. Late Ross and Starn) in San Joaquin County, California. Wood cankers and discoloration also occurred in branches, generally initiating at pruning wounds. Approximately 8 symptomatic twigs or branches per orchard were collected to proceed with the isolation of necrotic tissues on acidified potato dextrose agar (APDA). Isolations consistently yielded colonies of the fungal pathogen Calosphaeria pulchella (Pers. : Fr.) J. Schröt. (Réblová et al. 2004; Trouillas et al. 2012). Pure cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. Colonies on APDA grew dark pink to red or purple in their center, with a white margin. Conidiogenesis was phialidic, producing round conidial masses at the tip of phialides. Conidia were produced abundantly on APDA, and were hyaline, allantoid to oblong-ellipsoidal, 4 to 5.5 (7) × 1.2 to 2.3 μm (n = 60). Two representative isolates (SJC-62 and SJC-64) were selected for genomic DNA extraction and sequencing of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers and the beta-tubulin (TUB2) gene region using primers Bt2a and Bt2b. Consensus sequences of the two genes for the two isolates (ITS: PP063990, PP063991; TUB2: PP068303, PP068304) were compared to reference sequences (Réblová et al. 2015; Trouillas et al. 2012) using BLAST analysis. The ITS sequences of SJC-62 and SJC-64 were 99.8 and 99.5% identical to that of C. pulchella ex-type strain CBS 115999 (NR145357) and reference strain SS07 (HM237297); the TUB2 sequences were at least 98.5% identical to that of C. pulchella CBS 115999 (KT716476). Pathogenicity tests were conducted in 2- to 3-year-old healthy branches on 7-year-old peach trees, cvs. Loadel, Late Ross and Starn using the two fungal isolates and a control treatment (1 branch per treatment and 3 branches per tree) on each of 8-tree replicates. Branches were inoculated in June 2023 following wounding with a 5 mm cork borer to remove the bark and placing an agar plug from the margin of 10-day-old colonies on APDA directly into the fresh wound. Sterile agar plugs were used as controls. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to retain moisture. The experiment was completed twice. After four months, cankers and vascular discolorations developed around the inoculation sites. Length of vascular discoloration in inoculated branches averaged 72, 75, and 79 mm, for the Loadel, Starn, and Late Ross cvs., respectively. Calosphaeria pulchella was re-isolated from inoculated branches at 80 to 100% recovery rate, thus fulfilling Koch's postulates. The average length of vascular discoloration in the control was 13.5 mm and no fungi were recovered from control branches. Calosphaeria canker caused by C. pulchella is a global disease of sweet cherry. Recently, it was reported to cause cankers in peach trees in Chile (Grinbergs et al. 2023). To our knowledge, this is the first report of C. pulchella causing cankers and twig dieback of peach trees in the United States. These findings improve our knowledge of the etiology of canker diseases affecting peach trees and is critical for the development of effective disease management strategies.

Anthracnose of Brachybotrys paridiformis Caused by Colletotrichum siamense in Northeast China.

Brachybotrys paridiformis Maxim. ex Oliv. (Boraginaceae) is a perennial medicinal plant and vegetable that is cultivated commercially in China. Anthracnose is a devastating disease of B. paridiformis, with annual production losses exceeding 33% based on our survey. In July 2021, anthracnose of B. paridiformis was observed on 2-year-old plants in Shenyang city, Northeast China, which is the most important region for B. paridiformis cultivation. Round or irregular-shaped black spots were exhibited on leaves, with the leaf edges most commonly infected. As the necrosis expanded, the leaves withered and dropped; young leaves were generally not infected (Fig. 1). More than 40% of the plants in a 21-ha sampling field were infected in 2021. Symptomatic leaves (n = 20) were collected and the diseased tissue was cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes. After a 3-day incubation in darkness at 25 °C, 18 suspected single-pure morphologically identical Colletotrichum isolates were obtained and sequenced. Isolate SQZ9 was randomly selected and identified. Colonies on PDA were initially white, but gradually became pale brownish with a reverse side that was pale yellowish to pinkish. Aerial mycelia were grayish-white, dense, and cottony, with microsclerotia detected on some aging mycelia. The detected single-celled conidia (11.65-17.25 × 4.25-6.15 µm; n = 50) were fusiform to cylindrical with obtuse to slightly rounded ends. Appressoria were ovoid to clavate and medium brown. Setae were not observed. The morphological characteristics were similar to those of Colletotrichum spp. (Prihastuti et al. 2009; Weir et al. 2012). Initial BLAST searches of the GenBank database revealed the SQZ9 rDNA internal transcribed spacer region (OP389109, 566 bp), glyceraldehyde-3-phosphate dehydrogenase (OP407730, 260 bp), chitin synthase (OP407731, 301 bp), calmodulin (OP407732, 712 bp), actin (OP407733, 282 bp), glutamine synthetase (OP407734, 909 bp), β-tublin (OP407735, 498 bp), and superoxide dismutase (OP407736, 396 bp) sequences were respectively 99%-100% similar to the C. siamense type strain JX010278, JX010019, JX009709, GQ856775, GQ856730, JX010100, JX010410, and JX010332 sequences (Carbone & Kohn 1999; Moriwaki & Tsukiboshi 2009; Stephenson et al. 1997). The SQZ9 identity was confirmed by constructing a phylogenetic tree combining all loci, which grouped the isolate and the C. siamense type strain in the same clade (Fig. 2). For pathogenicity tests, 15 healthy 2-year-old plants (3 plants per pot) were spray-inoculated with SQZ9 conidial suspension (1 × 105 conidia/mL) at 2 mL per plant. Same number of plants sprayed with water were used as control. This experiment was repeated twice. All plants were covered with clear plastic bags for 72 h to maintain high humidity and then placed in a greenhouse (29 °C, natural light, and 85% relative humidity). After six days, the inoculated leaves exhibited symptoms that were similar to those observed in the field, but the controls were symptomless. The same fungus was recovered from inoculated symptomatic leaves, and its identity was confirmed by sequencing and a phylogenetic analysis. This is the first report of C. siamense causing anthracnose on B. paridiformis in China. Future studies should assess the effectiveness of chemical and biological control measures for managing this disease.

First Report of Calonectria montana Causing Leaf Spot on Ligularia fischeri in China.

Ligularia fischeri (Ledeb.) is a perennial herbal plant of Compositae that is cultivated commercially in China as a medicinal, ornamental, and edible plant. Leaf spots were observed in 2-year-old L. fischeri in Benxi County of northeast China, in August 2021. Irregular reddish brown spots ranging from 3 to 11 mm were observed on infected leaves, and each leaf had dozens of spots (Fig. 1). As the disease progressed, the diseased spots withered and the centers fell out, and multiple lesions merge into large diseased spots, causing leaf wilting. The roots and stem bases were not infected during the reproductive stage. More than 37% of the plants in a 18 ha field were infected in 2021. The ten diseased leaves were collected and cut into small (3-5 mm) pieces, which were surface-disinfested by immersing into 1% NaOCl for 2 min and rinsing with sterile distilled water three times. The leaf pieces were then placed on acidified potato dextrose agar (PDA) in petri plates and incubated in the dark at 25°C. Twenty isolates with the same morphological characteristics were obtained. Isolates were further purified by starting a new colony for each isolate from a single spore collected from water agar. Isolate TYTW7 was randomly selected for identification and pathogenicity testing. It grew rapidly and produced profuse aerial mycelia with a carmine red underside. The conidiophores had many fertile branches and formed an elongated stipe with a sphaeropedunculate vesicle at the tip. The one-septate conidia were cylindrical and almost straight with parallel walls and rounded ends. Their sizes ranged from 30.35 to 51.76 × 2.93 to 5.01 μm (n = 100) and the pathogens were initially identified as Calonectria sp. (Crous 2002; Crous et al. 2004; Lombard et al. 2015, 2016). Further confirmation of the identification was determined according to published method (Liu and Chen 2017; Shao and Li 2021). The partial gene regions including the translation elongation factor 1-alpha (GenBank accession no. OP290551), histone H3 (OP290552), calmodulin (OP290553) and β-tubulin (OP290554) were obtained, and BLAST searches showed 99-100% homology with the ex-type culture CERC 8952 (MF527049, MF527065, MF527081 and MF527107) and phylogenetic analysis combining all loci revealed that the isolate TYTW7 and the type strain of Ca. montana clustered in one group (Fig. 2). Based on morphological characteristics and phylogenetic analysis, isolate TYTW7 was identified as Ca. Montana. Healthy 2-year-old plants were used for the pathogenicity test. A spore suspension (1×105 spores/mL water) was used to inoculate three host plants; sterile water was sprayed on the same number plants serving as a control. The experiment was repeated three times. All plants were incubated at 27±2°C (12h photoperiod) and were evaluated after seven days. The inoculated plants showed lesions on the leaf surface, similar to those in the field, and the control remained symptomless. The pathogens were successfully reisolated and identified by sequencing, and no pathogens were isolated from symptomless control plants. To our knowledge, this is the first report of Ca. montana causing L. fischeri leaf spot. The disease poses a threat to the production and more control strategies are needed on management options to minimize losses.

First Report of Diaporthe fruit rot and stem canker of hydroponic cucumber caused by Diaporthe sp. in the United States.

Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: D. cucurbitae, D. melonis, D. longicolla (syn. D. eres), D. pterocarpi, D. sclerotioides, D. sojae, and D. ueckerae (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers (Cucumis sativus) 'Katrina' and 'Alcazar' were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of 'Katrina' and 17% of 'Alcazar'). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel Diaporthe sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch's postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers 'Katrina,' grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber 'Katrina' were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.

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 Berkeleyomyces basicola Causing Black Shoot Rot of Raspberry in Nursery Production in the United States and Worldwide.

Commercial production of raspberry (Rubus ideaus) transplants is almost exclusively accomplished through clonal propagation. One system involves forcing young shoots to grow from roots. The shoots are cut and rooted in propagation trays and referred to as tray plants. Sanitation is important during tray plant production as this method carries some risk due to contamination by substrate pathogens. In May 2021, a new disease was observed on raspberry tray plant cuttings at one nursery location in California, and observed again in 2022 and 2023 but at a much lesser extent. Multiple cultivars were affected; however, up to 70% mortality was observed on cv. RH740.1. In less affected cultivars, mortality ranged from 5-20%. Symptoms included chlorotic leaves, lack of rooting, and blackening at the basal end of shoots, followed by death of the cutting. Affected propagation trays had inconsistent foliage and patchy growth. Chains of chlamydospores (two to eight spores in each chain) similar in morphology to Thielaviopsis species (Shew and Meyer 1992) were observed at the cut end of symptomatic tray plants using a microscope. Isolates were retrieved by incubating tissue on surface-disinfested (1% NaOCl) carrot discs in a humid chamber for 5 days until greyish black mycelium was observed (Yarwood 1946). Mycelium was transferred to acidified potato dextrose agar and formed a gray to black compact mycelial colony with both endoconidia and chlamydospores. Endoconidia were catenulate, single-celled with slightly rounded ends, colorless, and 10-20 µm x 3-5 µm in size; dark-colored chlamydospores were 10-15 µm x 5-8 µm in size. The ITS region of isolates 21-006 and 22-024 was amplified with ITS5 and ITS4 primers using a 48°C annealing temperature (White et al. 1990), Sanger sequenced (GenBank accession OQ359100) and yielded 100% match to Berkeleyomyces basicola accession MH855452. Pathogenicity was confirmed by dipping 80 grams of roots of cv. RH740.1 into a suspension of 106 conidia/mL of isolate 21-006 for 15 min. For the non-inoculated control, 80 grams of roots were dipped in water. Roots were then planted into trays of coir (Berger, Watsonville, CA). Six weeks after inoculation, twenty-four shoots were harvested from each treatment, stuck into propagation trays filled with coir and maintained in a humid chamber for 14-days to induce rooting. Tray plants were then harvested and assessed for root development, black basal shoot tips, and presence of chlamydospores. Forty-two percent of cuttings from the inoculated treatment had rotten basal tips and failed to root, in comparison to 8% of the cuttings from the non-inoculated control. Chlamydospores were visualized only on shoots that emerged from inoculated roots and B. basicola was isolated only from cuttings originating from inoculated roots. Post-inoculation isolates were confirmed as B. basicola using methods described above. To our knowledge, this is the first report of B. basicola infecting raspberry. Confirmation of this pathogen on tray plants is significant because of the potential impact this disease may have in commercial nursery production worldwide. In 2021, the value of the harvested raspberry crop in the U.S. totaled $531 M, of which California represented $421 M (USDA 2022). The value of the 2021 crop was highest in the U.S. ($531 M), followed by Russia ($512 M), Spain ($405 M) and Mexico ($332 M) (FAO 2021).

First Report of Botrytis californica Causing Gray Mold on Blueberry in China.

Blueberry (Vaccinium corymbosum) is a perennial shrub which grows in acid soil. Its cultivation area has recently expanded rapidly due to its unique flavor and high nutritional value (Silver and Allen 2012). In June 2021, gray mold symptoms (8 to 12% incidence) were observed on harvested fruits of the blueberry cultivar 'Lanmei 1' during storage in Jiangning (31°50'N, 118°40'E), Nanjing, China. The infection started with wrinkles, atrophy, and depressed spots on the fruit surfaces, ultimately resulting in fruit rot. To determine the causal agent, diseased fruits were sampled and rinsed with sterile water (Gao et al. 2021). Small fragments (5 × 5 × 3 mm) of decayed tissues were excised and plated onto acidified potato dextrose agar (PDA) (containing 4 ml of 25% lactic acid per liter). Plates were incubated at 25°C for 3 to 5 days, and edges of fresh culture were transferred onto new plates. This procedure was repeated thrice to obtain pure cultures. Two isolates (BcB-1 and BcB-2) were obtained. Their colonies appeared whitish to gray, with an average daily growth rate of 11.3 ± 0.6 mm (no. of plates = 30). Conidiophores were long and erect, 2560.9 to 4885.3 μm × 10.7 to 13.0 μm in size. Conidia were one-celled, elliptical to ovoid, nearly hyaline, and 9.6 to 12.5 μm × 6.7 to 8.9 μm in size. Sclerotia were gray to black in color and round or irregular in shape. These morphological features were identical to those of Botrytis spp. (Amiri et al. 2018). To further identify the isolates, we amplified four genetic markers including internal transcribed spacer region (ITS), heat-shock protein 60 (HSP60), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and DNA-dependent RNA polymerase subunit II (RPBII) (Saito et al. 2014; Walker et al. 2011). Sequences of BcB-1 and BCB-2 were deposited in GenBank under accession nos. OP721062 and OP721063 for ITS, OP737384 and OP737385 for HSP60, OP746062 and OP746063 for G3PDH, and OP746064 and OP746065 for RPBII, respectively. BLAST analysis indicated that these sequences shared high identities (99 to 100%) with those of other B. californica isolates. Phylogenetic analysis showed that BcB-1 and BcB-2 clustered with several reference isolates in the B. californica clade. To confirm their pathogenicity, fresh blueberry fruits were surface sterilized with 0.5% sodium hypochlorite, rinsed with sterile water, air-dried, then wounded thrice per fruit using a sterile needle at the equator. Twenty wounded fruits were sprayed with 10 ml conidial suspension (1 × 105 conidia/ml) of each isolate onto the fruit surface. Twenty fruits treated with sterile water were used as controls. Inoculated or non-inoculated fruits were incubated at 25°C with 90% relative humidity. The pathogenicity test was performed twice. After 5 to 7 days, all inoculated fruits developed disease symptoms similar to those observed on the original fruits, while non-inoculated control fruits were asymptomatic. Re-isolated pathogens from the inoculated fruits exhibited identical morphological characteristics to those of BcB-1 and BcB-2. Their identity as B. californica was also verified based on their ITS sequences. Previously, B. californica has been reported to cause gray mold on blueberry in the Central Valley of California (Saito et al. 2016). To our knowledge, this is the first report of B. californica causing gray mold on post-harvest blueberry fruits in China. These results can provide the basis for future studies on the occurrence, prevention, and control of this disease.

First Report of Colletotrichum graminicola Causing Maize Anthracnose in Galicia, Northwestern Spain.

Maize (Zea mays) is one of the most important crops worldwide, and fungal diseases are responsible for major losses in food production. Anthracnose caused by Colletotrichum graminicola can infect all maize tissues, although stalk rot and seedling blight cause more significant economic damage (Munkvold and White, 2016). Anthracnose stalk rot is characterized by a distinctive external blackening of the lower stalks resulting in large black streaks, while the pith turns dark brown and has a shredded appearance. Like most stalk rots, the most obvious symptom is a sudden death of plants before grain maturity, and plant lodging. Symptoms commonly appear late in the season, suspicious maize stems of cultivar Tuy exhibiting symptoms of anthracnose stalk rot were collected from a field in Pontevedra, Galicia, Spain (Geographical coordinates: 42°23'27.1" N - 8°30'46.3" W) between June and December of 2022. Stem samples, approximately 50 mm2, were dissected and surface-disinfected for 90 seconds in 20% sodium hypochlorite (v/v) and rinsed three times in sterile distilled water. The samples were transferred to one half-strength acidified potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and lactic acid 90% (1.5 mL/L) and incubated for 5 days at 25 ºC (Sukno et al. 2008). Single spores were transferred to fresh PDA plates to obtain pure culture isolates. A total of six isolates were obtained, and among them, two were selected for further characterization (SP-36820-1 and SP-36820-3). Colonies grown on PDA have dark gray aerial mycelium with orange-colored spore masses. Conidia are falcate, slightly curved, tapered toward the tips, and are produced in acervuli with setae, measuring 37.65 to 24.84 x 8.02 to 4.67 µm, respectively (n = 100). These morphological characteristics are in agreement with C. graminicola previously described by Bergstrom and Nicholson (1999). Isolates were grown in potato dextrose broth (PDB) for 3 days at 25 ºC and total genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA, USA). The internal transcribed spacer region of rDNA and the manganese-type superoxide dismutase gene (SOD2) were amplified using primers ITS4/ITS5 (White et al. 1990) and SOD625/SOD507 (Fang et al. 2002) and consequently sequenced. GenBank BLAST analysis revealed that the sequences were 100% identical to strains of C. graminicola. All sequences were deposited in GenBank (see e-Xtra 1 for accession numbers). To confirm Koch's postulates, plants of a derivative of maize inbred line Mo940 (developmental stage V3) were placed horizontally in a tray for inoculation and 20 droplets (7.5 µL) of a suspension of 3 x 105 conidia per milliliter were placed on the surface of the third leaf. The trays were closed to retain moisture and incubated overnight at 23ºC. The next day, the plants were returned to a vertical position and incubated in a growth chamber at 25ºC with 80% humidity and a light cycle of 16 h of light and 8 h of dark (Vargas et al. 2012). After four days inoculated leaves presented brown elongated lesions with necrotic centers consistent with C. graminicola infection, whereas control plants remained asymptomatic. The strains reisolated from infected leaves were morphologically identical to the original isolates. To our knowledge, this is the first report of Colletotrichum graminicola causing maize anthracnose in Spain. Recently, maize anthracnose was also reported in Bosnia and Herzegovina and China (Duan et al. 2019; Cuevas-Fernández et al. 2019), suggesting the pathogen's geographic range is increasing, which may be a threat to maize cultivation in locations with optimal humid conditions for disease development.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp (Cannabis sativa) in Missouri.

Charcoal rot, caused by Macrophomina phaseolina, is abundantly present in the soil and has been reported as pathogenic to both soybean and corn, as well as numerous other hosts, including hemp grown for fiber, grain, and cannabinoids (Casano et al. 2018; Su et al. 2001). Hemp (Cannabis sativa) production in Missouri was a relatively new addition to the 2021 growing season. Charcoal rot was reported in Reynolds, Knox, and Boone counties in Missouri from commercial and experimental fields. One of the fields in question experienced heavy disease pressure and had an uneven stand loss, but the total loss was estimated at approximately 60% of the field and was attributed to charcoal rot. Charcoal rot signs and symptoms, microsclerotia on the lower stem and root tissue, wilting and stem discoloration, were observed on a majority of the hemp plants received at the University of Missouri Plant Diagnostic Clinic in July and late Fall of 2021, including samples from Bradford Research Farm in Boone County and Greenley Research Center in Knox County. Root and crown tissue from the hemp plants from the Greenley Research Center were cultured onto acidified potato dextrose agar (APDA). Macrophomina phaseolina and other fungi grew from the plated tissue after about three days of incubation at room temperature. Macrophomina phaseolina was confirmed based on the presence of melanized hyphae and microsclerotia (Siddique et al. 2021). The microsclerotia were black, round to ovoid shaped and ranged from about 34-87 µm (average 64 µm) in length and 32-134 µm (average 65 µm) in width (n = 44). A single-hyphae isolation from a putative M. phaseolina isolate was conducted to obtain a pure culture. The M. phaseolina culture from the Greenley Research Center was used to complete Koch's postulates of charcoal rot on four hemp cultivars. Sterilized toothpicks were added to pure cultures of M. phaseolina on APDA and incubated at room temperature for one week to allow for colonization and for use in greenhouse inoculation. Four hemp cultivars (Katani, Grandi, CFX-2, and CRS-1) were grown in a sterilized silt loam for three weeks in a greenhouse. About four plants per cultivar were grown for inoculation and one plant per cultivar was used as a control. The plants were inoculated with the M. phaseolina colonized toothpicks that were gently rubbed onto stem tissue and subsequently inserted into the soil at the stem. For six weeks, the plants were kept in greenhouse conditions of 25°C with a 12-hour light and dark cycle and were watered when soil appeared dry. Plants were kept in a loosely sealed container constructed from wood and vinyl sheeting to minimize cross contamination with other plants grown in the same greenhouse. Plants were monitored weekly for charcoal rot symptoms. Symptoms that resembled charcoal rot, wilting and microsclerotia on the lower stem, were present on inoculated plants after about four weeks and symptoms were not present on the control plants. Isolates resembling M. phaseolina in culture were recovered from symptomatic plants; therefore, Koch's postulates were successfully fulfilled and the fungus was recovered from the inoculated plants. DNA was extracted from the pure cultures of both the initial isolate and the isolate obtained from Koch's postulates using GeneJet Plant Genomic DNA Purification Kit (Thermo Scientific, California, USA) and the internal transcribed spacer (ITS) region of ribosomal DNA including ITS1, 5.8S, and ITS4 regions were amplified using universal primers ITS1 and ITS4 (White et al. 1990). The ITS region was sequenced and compared to reference sequences in GenBank by BLAST analysis. Recovered isolates (GenBank accession no. OQ455934.1) showed closest sequence similarity (100%) to M. phaseolina accession number GU046909.1. Little is known about the life cycle, growth conditions, and possible inoculum buildup in the soil in hemp in Missouri. In addition, M. phaseolina is a known pathogen of corn and soybean and effective management strategies are challenging for these crops as well due to the broad host range of the pathogen. Cultural management practices, such as crop rotations to reduce inoculum in the soil and closely monitoring for symptoms, may help reduce the severity of this disease.

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 Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

First report of Fusarium equiseti causing root and crown rot in tomato in Mexico.

Tomato (Solanum lycopersicum L.) is one of the most important crops in Mexico due to its economic and nutritional value. Among the main diseases in tomato production is Fusarium wilt, which can cause 60% production losses (Ascencio et al, 2008). Mixed infections of Fusarium species or other fungi genera, would increase disease severity. During April to May of 2021, tomato plants with more than 60 days old, were collected from the main production areas of Aguascalientes (22°03'46.5"N 102°05'17.4"W and 22°04'53.64"N 101°58'55.81"W) and Zacatecas (23°05'59.2"N 102°41'07.3"W and 22°16'52.1"N 102°00'11.8"W) Mexico states. Plants showed main root rot, vascular bundles necrosis with corky appearance, stem crown rot, and ascending yellowing. The main root and stem crown were cut in 0.25 cm2 pieces and disinfested in 2% NaClO for one minute, rinsed with distilled water two times, placed on acidified potato dextrose agar (PDA) medium, and incubated at 25 ± 2°C for 7 days. Characteristic Fusarium growths were purified by hyphal tip on PDA, subsequently pure strains were obtained by single-spore isolation method. Several fungi colonies were obtained, but we focused on the colonies that showed abundant aerial mycelium of white color and irregular growth, which turned yellowish to golden and brown color as it ages. Carnation leaf agar (CLA) medium were used for conidia and sporodochium development. Chains of terminal, intercalary and agglomerated chlamydospores with thick, rough brown walls of 18.9 (7.46) µm in diameter (n=120) were observed in the mycelium. Macroconidia with 5 to 7 septa were 30 to 75 (28.32) µm in long and 1.2 to 4.8 (3.2) µm in wide (n=72). Basal cell developed in foot-shape, apical cell was elongated and slightly curved, and some macroconidia had swollen midd-cell. Sporodochium was orange to brown in color and microconidia were absent (Figure 1). Two representative strains from each state, LCA-3.1 and EMA-1 from Aguascalientes and ECZ-4 and LRZ-6 from Zacatecas, were selected for DNA amplification of ITS, TEF-1α and RPB2 regions, with universal primers ITS1/ITS4, EF1/EF2 and 2-5F2/7cR (White et al.1990; O'Donnell et al. 1998, 2013). PCR products were sequenced by Psomagen, Inc. (USA). The sequences obtained showed 100% of similarity among themselves and within species of the Fusarium incarnatum-equiseti species complex (FIESC) with nucleotide NCBI accessions NR_121457 (Type material) for ITS and MW362069 for TEF-1α; and 99.28% with MN170399 for RPB2 in FUSARIOID-ID database. According to morphological (Leslie and Summerell, 2006) and molecular characteristics, isolates were identified as Fusarium equiseti (FIESC 14). The LCA-3.1 sequences were selected to be deposited in GenBank with accession numbers OM812801 (ITS), OM937108 (TEF-1α) and ON653596 (RPB2). Pathogenicity tests were performed twice, under greenhouse conditions in tomato seedlings of cv. Rio Grande. Five tomato seedlings were inoculated by root immersion method (Lopez et al, 2018) in a 1x106 spores/mL solution for 8 min, and transplanted to 1L pots with sterile peat. Five controls plants were immersed in sterile water. At 14 days after inoculation, a general plant decline and slower growth compared to the control plants were observed. Subsequently, plants showed root rot, vascular necrosis, and a brown ring in stem crown. Controls were symptomless. The fungi were re-isolated from symptomatic plants and were morphologically similar to the inoculated strains. Patel et al. (2017) described the pathogenic and toxic effects of F. equiseti on tomato, causing low seed germination, and low root and shoot growth. This is the first report of F. equiseti causing root and stem rot in tomato plants in Mexico.

First report of Fusarium proliferatum causing leaf spot on tea plants in Jiangxi Province, China.

Tea (Camellia sinensis (L.) O. Kuntze), a perennial evergreen shrub, is one of the most important cash crops in China. In September 2021, leaf spot symptoms were observed on approximately 30% of tea plants in a 2 ha commercial field of Lushan (29°33'0" N, 115°58'48" E), Jiangxi Province, China. The symptoms initially appeared as small, gray lesions, and later became larger (10-15 mm in diameter) circular to irregular spots with light brown centers and gray borders. To isolate the pathogen, small pieces (3×3 mm) cut from the margins of lesions were sterilized with 75% ethanol for 10 s, 0.1% HgCl2 for 20 s, and then rinsed three times with sterile water. The pieces were placed onto acidified potato dextrose agar (APDA) plates, and incubated in darkness at 28℃. Pure cultures were prepared by subculturing hyphal tips. A total of 16 fungal isolates were obtained, and the colonies of 15 isolates (isolation rate 93.8%) looked identical, resembling those of the genus Fusarium. The colonies were white to pink with purple woolly mycelium. After 10 to 15 days incubation, slightly curved macroconidia with three to four septa measuring 14.0 to 34.5 × 2.0 to 3.5 µm (n = 50), and oval, unicellar microconidia measuring 4.0 to 9.0 × 1.5 to 3.5 µm (n = 50) were observed. These morphological characteristics were similar to that described for Fusarium proliferatum (Leslie and Summerell 2006). Genomic DNA of representative isolates (LSZWY, LSZWY2, LSZWY3) was extracted with the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd, Shanghai). The translation elongation factor 1 alpha gene (EF-1ɑ) was amplified using primers EF-1H / EF-2T (O'Donnell, et al. 2015). PCR product was sequenced and the sequence was 709 bp (Accession No. OL614004, ON357634, ON595710). BLAST search results showed that it had 99.9% identity with the EF-1ɑ gene sequence of F. proliferatum (MH341215, MT371378). To test pathogenicity, nine leaves from 5-year-old healthy tea plants (Ca. Luyun 3) were wounded using a sterilized needle and inoculated with a 20µl conidial suspension (2 × 107 conidia·mL-1) on one side of the plants and the other side with sterilized distilled water as a control. All leaves were incubated in a growth chamber at 28℃ and 80% relative humidity with a 12 h light/dark photoperiod. Seven days later, all inoculated treatments showed symptoms identical to those observed in the field, while the control remained asymptomatic. The experiment was repeated three times with similar results. Koch's postulates were fulfilled by successful re-isolation and morphological and molecular identification of F. proliferatum from the inoculated leaves. This pathogen can cause diseases of many crops, e.g. tobacco, Polygonatum cyrtonema and others (Li, et al 2017; Zhou, et al. 2021). However, this is the first report of F. proliferatum causing leaf spot on tea plants in China. This new disease poses a threat to the yield and quality of tea and methods need to be developed for its control and to prevent further spread.

First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States.

Perilla mint (Perilla frutescens (L.) Britt.) is an annual plant native to Asia and considered invasive in North America where it has escaped cultivation as an ornamental (Miller 1947; Swearingen et al. 2010). In August 2021, an anthracnose disease was observed on invasive perilla found along the disturbed margins of a forest in Frederick County, Maryland, United States. Symptoms included necrotic lesions with chlorotic halos, were concentrated in the lower canopy, and caused premature defoliation of lower leaves (Figure S1). Leaves from four plants were surface sterilized by rinsing for 30 s in 70% ethanol, 60 s in 0.8% NaClO, and 60 s in sterile water and then incubated on 2% water agar under ambient laboratory conditions to permit sporulation. After three days, spores that exuded from individual lesions were streaked onto acidified potato dextrose agar. Two single-conidium isolates were recovered from each plant. All eight isolates were identified to species using DNA sequences. A single isolate (21-067) was selected at random for morphological characterization and completion of Koch's postulates. Morphological features were recorded after seven days of growth on synthetic low-nutrient agar (SNA) and potato dextrose agar (PDA) incubated at 22°C under 12 hr UV-B and white fluorescent lighting. Measurements were based on a minimum of 20 observations per structure. Cultures on SNA were flat, hyaline to pale salmon, lacked sporodochia and grew at a rate of 1.3 mm day-1 (n = 3). Vegetative hyphal width was (minimum-maximum) 1.5-4.0 μm, (average ± standard deviation) 2.7 ± 0.9 μm. Conidiophores arose directly from hyphae, were hyaline, smooth, unbranched and measured 40.0-180.0 x 1.5-4.5 μm, 102.3 ± 33.9 x 2.7 ± 0.8 μm. Conidia were single celled, straight, hyaline, glabrous, rounded at both ends and measured 10.0-20.0 x 3.8-6.3 μm, 15.4 ± 2.5 x 4.9 ± 0.7 μm. Setae, observed only on PDA, were pale brown, 1-2 septate, straight, blunt tipped and measured 42.5-97.5 μm, 70.8 ± 13.4 μm. Appressoria formed on PDA were single-celled, pigmented, smooth, and obovoid with entire margins, measuring 5.2-7.7 x 8.6-13.8 μm, 6.8 ± 0.6 x 10.8 ± 1.4 μm. These characteristics were consistent with members of the Colletotrichum destructivum species complex. Partial DNA sequences from five loci were amplified following the procedures of Damm et al. 2014: internal transcribed spacer region of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and beta-tubulin (TUB2). Pairwise sequence comparisons to references using the Blastn algorithm found 99.8% similarity between isolate 21-067 and ex-type C. shisoi isolate JCM 31818: MH660930 (ITS, 545/546 bp), MH660931 (GAPDH,192/192 bp), MH660929 (CHS-1, 278/280 bp), MH660928 (ACT, 262/262 bp), and MH660932 (TUB2, 511/511 bp) (Altschul et al. 1990; Gan et al. 2019). Subsequently, sequences from all eight isolates were aligned with Clustal Omega (Sievers and Higgins 2018), concatenated, and used in a Bayesian phylogenetic reconstruction (Huelsenbeck and Ronquist 2001) of the C. destructivum species complex (Figure S2), confirming the species identity as C. shisoi. Sequences were deposited in NCBI GenBank under accession numbers OM865277-OM865284 and OM885059-OM885090. Koch's postulates were fulfilled using perilla grown in a greenhouse until second true leaves emerged. Inoculum washed from two-week-old fungal cultures grown on potato dextrose agar was adjusted to 4 x 104 conidia mL-1 and applied to three replicate plants with an aspirator until runoff. Three plants were sprayed with sterile water as a negative control. Plants were covered with plastic bags for 72 hrs and maintained in a growth chamber at 20°C and 80% RH for 14 days. Inoculated plants displayed disease symptoms similar to those observed under field conditions, and control plants did not develop symptoms. Colletotrichum shisoi was reisolated from symptomatic tissue and reidentified based on morphology. The experiment was completed twice. To the authors' knowledge, this is the first report of C. shisoi on P. frutescens in the United States. Colletotrichum shisoi has not been reported as a pathogen on other plants in the United States and may have potential use as a biological control agent for invasive perilla.

First Report of Didymella rhei causing leaf spot on rhubarb in New York.

First Report of Didymella rhei causing leaf spot on rhubarb in New York E. J. Indermaur1, C. T. C. Day1, and C. D. Smart1† 1School of Integrative Plant Science, Section of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva NY 14456 †Corresponding author: C. D. Smart; Email: cds14@cornell.edu Rhubarb (Rheum spp.) is a perennial grown across the northern United States for petiole production (Foust & Marshall 1991). In August 2021, leaf spots were observed on rhubarb growing in a two-acre field in Erie Co., NY (Fig. S1). Approximately 30% of the plants in the field had leaf spot with disease severity of 5%. Initial symptoms on leaves were light brown, circular lesions with red margins that later coalesced into irregular spots. Lesion centers were dry with concentric rings, often perforating as they enlarged. Lesions on petioles were light brown, fusiform, and sunken with red margins. To identify the causal agent(s), symptomatic leaves and petioles from 50 plants (cultivar unknown) were collected with a W-shape sampling scheme. Lesion margins were surface sterilized with 70% ethanol for 60 s, 10% bleach for 60 s, rinsed in sterile water, plated on acidified potato dextrose agar (PDA), and incubated for two to four days at 20˚C. Hyphal tips from colony edges were transferred to new PDA plates. After 20 days, colonies (n=53) were olivaceous buff to grey olivaceous, producing white to grey, sparse aerial mycelium. Brown to black pycnidia were produced within five days in concentric rings around plate centers. Pycnidia were globose to subglobose, with one to two non-papillate or slightly papillate ostioles, and with mean diameter 75.8 (30.8 to 113.5) µm (n=20). Conidia were hyaline, ellipsoid or allantoid, and aseptate with mean ± SD dimensions of 6.2 ± 0.4 (4.9 to 8.1) x 2.2 ± 0.4 (1.3 to 3.3) µm (n=30) (Fig. S2). Based on these morphological characteristics, the isolates were initially identified as Didymella rhei [Ellis & Everh] (Qian Chen & L. Cai) (Boerema 2004). To confirm the identity, mycelia were scraped from PDA plates and homogenized using a TissueLyser II (Qiagen Inc.). Genomic DNA was extracted with a DNeasy Plant Mini Kit following manufacturer's instructions (Qiagen Inc.). PCR assays with primers ITS 4 and ITS 5 and fRPB2-7cR and RPB2-5F2 (Liu et al. 1999; Sung et al. 2007) were used to amplify the internal transcribed spacer (ITS) and the rpb2 gene regions of one representative isolate (strain RHU21204). Products were sequenced using Sanger chemistry. The sequences were deposited in GenBank with accession numbers OM903952 (ITS) and OM925897 (rpb2). The ITS and rpb2 sequences exhibited 99% (492/494 bp) and 100% (846/846 bp) identity with D. rhei accessions KF531831.1 and KP330428.1, respectively. Based on morphological and molecular characteristics, the pathogen was identified as D. rhei. To fulfill Koch's postulates, healthy leaves and petioles of four rhubarb seedlings (cultivars unknown) were spray-inoculated with a conidial suspension (1 × 107 conidia/ml) containing 0.2% Tween-20 from strain RHU21204. A tween suspension with no conidia was used as a control. Each treatment had three replicates. After inoculation, plants were placed in a 19˚C growth chamber with a 12-h photoperiod and misted for 30 min twice daily to maintain humidity above 80%. Initial symptoms were observed five days post inoculation (dpi), while control plants were asymptomatic. The pathogen was isolated 21 dpi from inoculated leaves and petioles with symptoms as described above (Fig. S1) and identified morphologically and molecularly as D. rhei. A representative isolate was deposited in the Cornell Plant Pathology Herbarium as CUP-070923. To our knowledge, this is the first report of D. rhei causing rhubarb leaf spot in New York and reducing the health and marketability of its host. Funding Source This project was funded by the College of Agriculture and Life Sciences, Cornell University. Literature Cited Boerema, G. H. et al. 2004. CABI Publishing. 288. Foust, C. M. and Marshall, D. E. 1991. HortScience 26:1360. DOI: 10.21273/HORTSCI.26.11.1360 Liu, Y. J. et al. 1999. Mol. Biol. Evol. 16:1799. Sung, G. H. et al. 2007. Mol. Phylogenet. Evol. 44:1204. DOI: 10.1016/j.ympev.2007.03.011.

Anthracnose of Tribulus terrestris Caused by Colletotrichum truncatum in Northeast China.

Tribulus terrestris L. is an annual herbaceous medicinal plant of Zygophyllaceae, which is cultivated commercially in China. Subrotund or irregular gray, sunken, necrotic spots ranging from 2 to 9 mm were observed on diseased leaves of T. terrestris landrace in Fushun County, Liaoning Province of northeast China in July 2021, with more than 32% of the plants being infected in a 18-ha field. The symptoms first appeared on older leaves and gradually spread to younger leaves. The lesions developed a white center gradually and became perforated; multiple lesions could coalesce (Fig. 1). Ten symptomatic leaves were collected and the diseased tissues were cut into small pieces, immersed in 1% NaOCl for 2 min, rinsed three times with sterile water, and placed on acidified potato dextrose agar (PDA) in Petri dishes at 25°C in darkness. Fifteen suspected Colletotrichum single-spore fungal isolates (JL1 to JL15) with consistent morphological characteristics were obtained, and isolate JL6 was selected for identification and pathogenicity testing. Colonies on PDA were flat with an entire margin, dense and white at first, then became dark gray with numerous black microsclerotia and formed a concentric circular pattern with aging. Conidia were single-celled, sickle-curved with a tapered tip and truncate base, ranging from 16.46 to 20.26 μm in length and 2.81 to 3.96 μm in width (n=100). Setae were dark brown, septate, straight with a slightly acute tip, 75.45 to 135.63×3.19 to 4.95 μm in size. Appressoria were dark brown, round or irregular, mostly in groups. All characteristics were consistent with the descriptions of C. truncatum (Damm et al. 2009). Further confirmation of the identification was determined according to methods described previously (Damm et al. 2009). The rDNA internal transcribed spacer region (OP364400, 585 bp), and actin (OP380867, 290 bp), beta-tubulin (OP380868, 498 bp), chitin synthase 1 (OP380869, 277 bp), glyceraldehyde-3-phosphate dehydrogenase (OP380870, 280 bp), and histone (OP380871, 411 bp) genes were amplified by PCR and sequenced (Carbone and Kohn 1999; Glass and& Donaldson 1995; Guerber et al. 2003; O'Donnell and Cigelnik 1997). BLAST results showed 98-100% similarity at 85-97% coverage compared to the corresponding sequences of the type strain CBS 151.35 (GU227862, GU227960, GU228156, GU228352, GU228254, and GU228058). Phylogenetic analysis combining all loci revealed that the isolate JL6 and the type strains of C. truncatum clustered in one group (Fig. 2). One-year-old healthy seedlings of T. terrestris (cultivar: landrace) were used for pathogenicity test. Suspension (1×105 conidia/mL) of isolate JL6 was sprayed on ten seedlings, and ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. All plants were kept at 28±1°C (12 h photoperiod), and were evaluated after 7 days. The inoculated plants showed lesions on the leaf surface, similar to those in the field, and the control remained symptomless. The pathogen was successfully reisolated and identified using the methods mentioned above. To our knowledge, this is the first report of C. truncatum causing anthracnose on T. terrestris, which will provide valuable information for designing strategies to manage anthracnose on T. terrestris.