First Report of Strawberry Anthracnose Crown Rot Caused by Colletotrichum siamense in New England.

In the summer of 2023, the Connecticut Agricultural Experiment Station was contacted by a farm in southern Connecticut due to reports of strawberry (Fragaria × ananassa) plants showing signs of severe wilting and crown rot across multiple fields, covering ~20 hectares. Cut crowns from diseased plants had marbled red and white lesions typically associated with anthracnose crown rot (ACR). Symptomatic plants were collected from five June-bearing cultivars (cvs. AC Valley Sunset, Lyla, Dickens, and Allstar) spanning four non-adjacent fields with incidence ranging from 5-90% and severity ranging mild wilting in low incidence fields to severe wilting/mortality in high incidence fields. Internal tissue from diseased crowns was surface sterilized in 0.6% NaOCL for 3 minutes, rinsed with sterile water, and plated on potato dextrose agar. After one-week, hyphal tips of fungi were transferred to fresh plates which formed dense mycelial mats of fluffy, greyish-white hyphae. Orange spore masses formed near the center of the colonies, each of which contained numerous cylindrical and fusiform straight conidia, matching spores within the genus Colletotrichum (De Silva et al. 2019). Average conidia (n=192) length was 15.7 ± 1.6 µm and width was 5.4 ± 0.7 µm. Fungi matching this morphology were isolated from 83% of the collected symptomatic crowns and hyphae were collected from two isolates, CT5-1 and CT23-1, for DNA extraction using the GeneJET Plant Genomic DNA Purification Kit. PCR was performed using primers targeting actin (ACT), calmodulin (CAL), ß-tubulin (TUB2), GAPDH (gpdA), and ITS, followed by Sanger sequencing, which yielded identical sequences for both isolates (CT5-1 Accessions numbers: PP002078-81, OR999066)(Carbone and Kohn 1999; Hassan et al. 2018; Templeton et al. 1992). These were combined with sequences from fourteen Colletotrichum genomes, all of which were aligned, trimmed, and concatenated using Mega11 (Tamura, Stecher, and Kumar 2021). Model selection was conducted using IQ-TREE and selected parameters were used to generate maximum-likelihood trees from all five loci individually and the concatenated sequence, all of which placed the isolates in a high confidence cluster with Colletotrichum siamense (Nguyen et al. 2015). To confirm the pathogenicity of the pathogen, strawberry plants (cv. Jewel) (n=5) five weeks after bare root transplant were infected. The base of each crown was penetrated 5 mm deep with a sterile 20 µL pipette tip and then inoculated with 10 µL of spores at a concentration of 106 spores/mL. Control plants (n=5) were inoculated with 10 µL of sterile water. Plants were maintained at 30°C day (16-hour)/20°C night (8-hour) in a growth chamber and assessed after 14-days. Four of the five inoculated plants had visible wilt symptoms and bisected crowns revealed the marbled red and white lesions typifying ACR. Control plants had no clear wilting and bisected crowns were visibly healthy. C. siamense re-isolated from infected tissue presented with identical hyphal /spore morphology and ITS/Tub2 were re-amplified and sequenced, yielding identical sequences to CT5-1. Plant inoculations with the same variety were repeated, yielding identical symptom development and crown lesions. C. siamense has been a dominant source of ACR throughout the southeastern US but has not previously been a major problem in the Northeast. Given the extent of the field infection, it is likely that these isolates can survive the colder winter temperatures of New England, but further experimentation is necessary to determine the extent of the pathogen's winter hardiness.

First Report of Neopestalotiopsis spp. Causing Leaf Spot and Petiole Blight on Strawberry in New England.

In October 2023, a Connecticut grower contacted The Connecticut Agricultural Experiment Station about a field of strawberry plants (Fragaria × ananassa) (cv. Ruby June) showing symptoms of severe leaf spotting and visual wilting. Upon visiting the field, leaves had lesions with a diffuse black halo and a light brown center and wilting symptoms, which appeared driven by petiole lesions and presented as dark brown stripes with a reddish-purple halo. Symptoms were observed on 80 to 90% of plants within the block, nearly all of which (>90%) presented with both leaf spots and severe wilting. Diseased tissue was collected from 20 leaves and 25 petioles, sterilized in 0.6% NaOCL, and plated on potato dextrose agar. After hyphal tipping a morphologically identical fungus was isolated from 70% of leaves and 88% of petioles, which formed a dense white mycelial mat with moderate aerial mycelium and conidiomata that exuded dark brown conidial masses. The underside of the mycelial mat was yellowish. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate with a single basal appendage and 2-5 apical, matching the description of species within the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). The average conidia (n=74) length, not including appendages, was 29.9 ± 2.1 µm and the average width, at the widest point, was 7.5 ± 0.7 µm. Aerial hyphae were collected from two isolates, CT58-1 and CT62-2, and DNA was extracted for further molecular characterization. PCR was performed with primers targeting actin (ACT), ß-tubulin (TUB2), and ITS prior to amplicon sequencing (Carbone and Kohn 1999; Hassan et al. 2018). Sequences were queried against the NCBI whole genome shotgun database, and aligned sequences from 13 species (including Neopestalotiopsis, Pestalotiopsis, and Pseudopestalotiopsis) were collected for each locus. Sequences were aligned, trimmed, and concatenated using Mega11, and IQ-TREE was employed for model selection (Nguyen et al. 2015; Tamura et al. 2021). A maximum-likelihood tree placed the isolates in a high-confidence cluster with Neopestalotiopsis rosae, confirming this placement of these isolates within the genus (CT58-1 Accession #: PP715979-89; PP707735). To confirm pathogenicity, CT58-1 was grown on autoclaved strawberry leaves to induce sporulation, and a suspension of 105 spores/ml was made. Five milliliters of this spore suspension was sprayed on six 6-week-old strawberries (cv. Jewel), and water was sprayed on the same number of control plants. Plants were at 100% humidity for two days and then kept in the greenhouse for 3 weeks to observe symptoms. Inoculated plants presented with identical leaf spot and petiole lesions to field samples and no visual symptoms were observed on control plants. New isolations were made from infected petioles, which produced morphologically identical spores to those described above, and ITS/ACT loci sequencing yielded sequences identical to those of CT58-1. Spore production and plant inoculations were repeated with this new isolate, and identical symptoms were observed. This is the first report of Neopestalotiopsis infecting strawberries in New England and given the high disease incidence in the initial infected field and relative lack of disease in a neighboring field, it is likely that this pathogen was introduced on bare root plants. As the plants were sourced from a nursery in Ontario, Canada, it is likely that the pathogen is capable of overwintering in the Northeastern United States.

First Report of Globisporangium ultimum Causing Pythium Damping-off and Root Rot on Pepper in Guizhou, China.

Pepper (Capsicum annuum L.) is a popular vegetable and condiment consumed around the world. In the Guizhou Province of China, peppers are the most commonly grown crop on 300,000 planted hectares. A variety of diseases routinely occur on peppers in this province, resulting in yield losses (Liu et al., 2022). Root rot is one of the most common symptoms and produces poor root growth and wilting of pepper. In April 2023, symptomatic pepper plants displaying stunting, dwarfism, wilting, and root browning were collected from five fields in Guizhou, with disease incidence ranging from 10% to 20%. The collected rotten roots were cleaned with sterilize distilled water and placed in selective V8 juice agar (V8A) medium (15% clarified V8 juice with 2.5 g/L CaCO3 and 2% agar) containing nystatin, ampicillin, rifampicin, and miconazole, and incubated at 25℃ for 1 to 2 days (Morita and Tojo, 2007). Eight isolates with similar colony morphology were transferred to V8A medium via hyphal tipping, and incubated at 25℃ in the dark. Colony and sexual structures were observed using a microscope. Mycelium was aseptate and formed white cottony colonies. Globose, intercalary, or terminal hyphal swellings were observed with a diameter of 20.5 to 25 µm (average: 22 µm), and aplerotic oospores had a diameter of 15 to 20 µm (average: 17.5 µm) with a wall thickness of approximately 2 µm. Three representative isolates HSLJ-3, LJG-1, and LJY-2 were chosen for further molecular identification. Sequences of the internal transcribed spacer (ITS) and mitochondrial cytochrome c oxidase subunit 1 (cox1) genes were identified using primer sets ITS4/ITS5 (White et al., 1990) and OomCoxI-Levup/OomCoxI-Levlo (Robideau et al., 2011), respectively. All sequences were deposited in GenBank (accession nos. OR554005, PP083310, and PP083420 for ITS, and OR529247, PP093821 and PP093822 for cox1). BLAST analysis revealed all ITS and cox1 sequences exhibited 100% identity with Globisporangium ultimum (Pythium ultimum) isolate BR850 (GenBank accession nos. HQ643892.1 and HQ708933.1 for ITS and cox1, respectively). Phylogenetic analysis was performed by the maximum-likelihood method on the CIPRES web portal (https://www.phylo.org/portal2/login!input.action, accessed on 9 January 2024). For pathogenicity tests, each isolate was cultured in V8A medium containing 50 autoclaved wheat seeds at 25℃ for 7 days. Budding pepper seedling (cv. Huaxi) was transplanted into a 0.4 L pot containing sterilized commercial potting mix (Seedling Cultivation Substrate, Hunan Xianghui Agricultural E-commerce Co., Ltd.) which was saturated with deionized water. Eight infected and non-infected wheat seeds were placed near the roots of five pepper seedlings, respectively. Plants were placed in an artificial climate chamber, with a 14 h photoperiod and approximately 75% relative humidity at 25℃. After 14 days, inoculated seedlings showed symptoms of stunting, wilting, and rotting roots similar to those observed in the field. No disease was observed on the non-inoculated control plants. The pathogen was isolated from infected pepper roots and confirmed as G. ultimum by morphological and molecular analyses as previously described. This is the first report of G. ultimum causing root rot on pepper in Guizhou, China. This finding is critical to the discover of treatment options for this pathogen, thereby improving management practices to reduce yield losses in pepper.

First global report of Hyaloperonospora brassicae in commercial broccoli and cabbage microgreens.

Microgreens are a nutrient-dense enhancement to modern diets (Choe et al. 2018), whose small production footprint in protected systems facilitates rapid crop turnover and distribution to population centers. Eleven of the 25 most broadly grown microgreens are brassicas (Choe et al. 2018). In November 2023, kale, broccoli (H009B), and cabbage (H009C) microgreen crops in Michigan were observed with downy mildew, at disease severities of 3%, 40%, and 20% foliage on 10 x 16 cm seeded blocks of plants, respectively. These crops shared a germination chamber for at least three days, which was maintained at approximately 22℃ in very humid, dark conditions. Chlorosis and grayish, sunken necrosis characterized symptoms on cotyledon surfaces (Fig. 1). In humid conditions, thick, white-light gray sporulation was present on adaxial cotyledon surfaces, accompanied by sparse sporulation on abaxial surfaces and hypocotyls. Severely diseased plants were stunted and approximately 50% gradually succumbed to downy mildew. On microscopic examination, a Hyaloperonospora spp. was tentatively identified, with long sporangiophores that dichotomously branched 3 to 6 times and hyaline sporangia borne singly on flexuous terminal sterigmata (Fig. 2). Sporangia were round to oval, with average length of 23.1 (range 16.0 to 28.3) µm; width of 20.0 (15.0 to 25.6) µm; and average length:width of 1.2 (1.0 to 1.4); (n = 97 for all). Sporangia dislodged rapidly if disturbed or as humidity decreased. Two pathogenicity tests were initiated on two sequential days. Two cotyledons from originally infected broccoli and cabbage were suspended, abaxial-side down, on coarse mesh over an open 60-mm plate of pregerminated brassica seeds on a water-saturated filter, inside a sealed, clear plastic box. Boxes contained only one type of originally diseased host, with 15 to 20 seeds of transfer varieties in unique dishes. Boxes were incubated in the dark for 2 days at 19°C with a wet paper towel atop the cotyledons. Before removal, cotyledons were lightly brushed across the surfaces of the seedlings they were just suspended above. Seedlings were grown in boxes in the presence of indirect, ambient light for 9.5 hr/day for an additional 5 days before pathogen sporulation was apparent. Filter paper was resaturated as needed. Noninoculated control plants, maintained separate from inoculated plants, were asymptomatic throughout the experiments. Total disease incidence in transfer varieties was 43.5% of 'Graffiti' cauliflower, 18.7% and 15.7% of 'Nixon' and 'Blue Vantage' cabbage; 11.8% of 'Red Russian' kale, and 6.0% of 'Ironman' broccoli, combined from two experiments. All varieties listed had at least one plant successfully infected in both pathogenicity tests. Sporulation on transfer hosts was morphologically identical to originally affected crops. Sporangiophores and sporangia were removed from H009B broccoli and H009C cabbage plants using surface sterilized forceps, placed directly into DNA extraction tubes containing buffer CD1 (Qiagen PowerSoil Pro), then kit instructions were followed. Extracts were utilized as template for ITS and cox1 PCR amplification, using DreamTaq Mastermix and ITS4/6 (45 cycles; White et al. 1990) and Levup/Levlo primers (30 cycles; Robideau et al. 2011). Cycling conditions were as published, with the number of cycles indicated by primer set. Each reaction yielded a single amplicon of approximately 1000 and 700 bp, for ITS and cox1, respectively,. Amplicons were cleaned using ExoSap-IT and submitted for Sanger sequencing, using ITS6 and Levup as sequencing primers (Robideau et al. 2011; White et al. 1990). After quality trimming, amplicons shared >98.5% identity with H. brassicae (NCBI Genbank accession MG757792 or reference genome CANTFL010000892.1). Sequences were submitted to Genbank (PP093830, PP093831, PP776812, PP776813). This is the first report of downy mildew, caused by H. brassicae, in commercial brassica microgreens, crops with vast nutritional value and expanding production.

First Report of Lasiodiplodia theobromae Causing Fruit Crown Rot on Banana in Ecuador.

Post-harvest diseases like fruit crown rot (CR) on bananas (Musa spp.) worldwide are mainly attributed to Colletotrichum gloeosporioides (Berk. & Curt.) von Arx and Lasiodiplodia theobromae (Pat.) Griff. & Maubl (Sangeetha et al., 2012; Riera et al., 2019). In April 2019, at a banana farm (cultivar Williams) located in El Oro province (location at 79° 54' 05" W; 03° 17' 16" S), thirty hands were randomly collected from the postharvest process and further placed in a humid chamber at 20 ºC until signs of the disease progressed and became more evident (from 3 days to 20 days). Ten hands presented initial symptoms related to CR during the postharvest process, which included crown or peduncle rot with mycelial development on the crown's surface, leading to the blackening of tissues at the site of the wound left when the cluster was cut. Crown fruit fragments (~0.5 cm) from the edge of healthy tissue and diseased tissue underwent a series of disinfection steps, initially in ethanol (70%) for 1 min, followed by sodium hypochlorite (1%) for 1 min, rinsed three times with sterile distilled water, and dried on sterile filter paper for 10 min. The fragments were placed onto Potato dextrose agar (PDA) + chloramphenicol (100 mg L-1) and incubated at 25°C in darkness for five days. Five isolates with different colony morphologies were obtained. An initial screen of the pathogenicity of all isolates showed that only one isolate showed disease activity in banana crowns. This isolate, C1, showed grayish-white aerial mycelium in culture as described above and, after ten days, became black. We did a full pathogenicity test with C1 using ten individual banana fruits (cv. Williams Cavendish). Briefly, one disc (Ø of 5 mm) of the fungus with agar was placed on the acropetal part of the banana fruit (on the peel) and another piece in the crown without wounding. Inoculated fruit were in a humid chamber at 20 °C for 20 days. Uninoculated fruits constituted the control. Isolate C1 caused 100% of the fruit and crowns to rot, with symptoms similar to those initially observed from fruit collected at the postharvest process (Fig. S1d). The fungus was re-isolated from symptomatic tissue, and its identity was confirmed through morphological characteristics consistent with Lasiodiplodia sp. Matured conidia of all mono hyphal strains (Fig. S1b) appeared dark brown with a single septum, having an ovate shape, and displayed longitudinal striations along their thickened walls (Fig. S1c). The dimensions of the mature conidia ranged from 16.02 - 26.85 x 11.09 - 16.74 µm (n = 60). Morphological characteristics showed similarity to Lasiodiplodia sp. (Alves et al., 2008). Microscopic observations were further confirmed by sequencing three loci: the internal transcribed spacer (ITS), ß-tubulin, and partial translation elongation factor-1α (TEF-1α). Fungal genomic DNA from the C1 isolate was PCR amplified using ITS5/ITS4, EF1-728F/986R, and Bt2A/Bt2B primers, respectively, according to Glass & Donaldson (1995) and Bautista-Cruz et al. (2019). The resulting amplicons were sequenced, and those sequences were deposited in GenBank with the accession numbers ITS: PP532861, TEF-1α: PP551938, and ß-tubulin: PP537587. Sequence alignment was conducted using ClustalW under the MEGA 11.0 software package (Tamura et al., 2021). Subsequently, phylogenetic analysis was performed using Bayesian inference using the BEAST v1.8.4 program (Drummond & Rambaut, 2007). The concatenated sequence of the isolate revealed clustering to the Lasiodiplodia theobromae clade, confirming its identity. To our knowledge, this is the first report of this pathogen causing CR on banana fruit in Ecuador. Based on the report of CR in the country, banana exporters and the Ecuadorian government should consider developing disease management methods that include the cultivation, shipping, ripening, and storage processes of the fruit.

First Report of Anthracnose Caused by Colletotrichum siamense on Machilus thunbergii Siebold & Zucc. in South Korea.

Machilus thunbergii Siebold & Zucc., known as Japanese bay tree, is an evergreen tree distributed widely in East Asia, including South Korea, where the species is of ecological importance. Machilus thunbergii provides habitat for wildlife species and is a common urban tree. In September 2022, anthracnose symptoms on leaves were observed in Jeju (33°26'02.4"N, 126°19'48.8"E) and Tongyeong (34°49'27.1"N, 128°24'01.8"E) in South Korea. Disease incidence on leaves of each affected tree, naturally growing in an urban forest area covering approximately 0.5 ha was approximately ~ 70 % in each study area. Anthracnose symptoms that were observed on 70 to 80% leaves per tree in each study area included orbicular or irregular, whitish-grey spots on leaves that were 1.5 to 3.0 cm in diam. In some cases where leaves were severely affected, larger blotches were formed, leading to bleaching symptoms and eventually defoliation. For pathogen isolation, two or three leaves showing anthracnose symptoms from each of the 15 trees were randomly selected and brought to the laboratory. Fungal isolations were then directly made by transferring spores from acervuli that developed on diseased leaves onto potato dextrose agar (PDA) media. Cushion shaped acervuli filled with salmon to orange-colored conidial masses were produced on media approximately two weeks after the incubation at 25 ± 1°C with a photoperiod of 12 h. Conidia were single celled, hyaline, cylindrical with rounded ends, smooth walls, 13.7 to 18.1 µm long and 3.1 to 4.5 µm wide (n=30). Among 15 cultures that were successfully isolated, 10 isolates were retained based on culture characteristics, and two randomly selected monoconidial cultures were deposited in the culture collection (CDH) of the Chungnam National University, Republic of Korea (Accession No. CDH057-58). Two isolates selected, CDH057 and CDH058, were subjected to identification, and this was achieved based on multiplesequence comparisons using on internal transcribed spacer regions of rDNA (ITS1 and ITS2), partial sequences of actin (ACT) and ß-tubulin (TUB2) gene regions amplified using ITS1F / ITS4, ACT-512F / ACT-783R and T1 / Bt2b, respectively (Weir et al. 2012). The representative sequence data were deposited in GenBank under the accession numbers OR473277 and OR473278 for the ITS, OR480772 and OR480773 for ACT, and OR480774 and OR480775 for TUB2. The resulting sequences were further used for a phylogenetic analysis based on the maximum likelihood method using a concatenated dataset of the ITS, ACT and TUB2 gene sequences for Colletotrichum species in the C. gloeosporioides clade. The results showed that the pathogen isolated in this study clustered with Colletotrichum siamense (Vouchered specimens: MFLU 090230, COUFPI291, and COUFPI294) (Prihastuti et al. 2009). Sequence comparisons revealed that the isolates obtained in this study differed from the type species of C. siamense (MFLU 090230; FJ972613 for ITS, FJ 907423 for ACT, FJ907438 for TUB2) at 2 of 258 bp (∼0.8%) and 6 of 387 bp (∼1.6%) in the ACT and TUB2 sequences, respectively, while the ITS was identical to the type species. For pathogenicity tests, a total of ten three-year-old seedlings of M. thunbergii were used. The leaves of each tree were sprayed with 5 ml of conidial suspension (105 conidia/ml, isolate CDH057). Three control plants were sprayed with sterile water. After being sprayed, treated areas were sealed with a plastic bag for 24 hours to preserve humidity. Anthracnose symptoms, identical to those observed in the field, appeared five to seven days after the inoculations, while no symptoms were observed on control plants. The isolates used in the pathogenicity test were reisolated from 90% of lesions, and their identity was confirmed based on sequence comparisons, thus fulfilling Koch's postulates. Species of the C. gloeosporioides species complex include important plant pathogens, particularly C. siamense, which cause significant losses of economic and ecological relevance on a wide range of hosts (~ 100 hosts) (Talhinhas and Baroncelli 2021). Although C. fioriniae in the C. acutatum species complex, was found on M. thunbergii in South Korea (Thao et al. 2023), anthracnose associated with C. siamense on M. thunbergii has not been reported in the country. In this regard, this is the first report of anthracnose caused by C. siamense on M. thunbergii in South Korea. To effectively control the disease, more attention should be paid on the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.

First Report of Golovinomyces bolayi Causing Powdery Mildew on Lettuce in Mexico.

In August 2022, powdery mildew symptoms were detected on lettuce (Lactuca sativa) in a commercial field located in Quecholac, Puebla, Mexico. Signs appeared as whitish powdery masses on leaves. Disease incidence was about 100% and signs covered up to 40% of leaf surface. Mycelium was amphigenous forming white patches. Hyphal appressoria were indistinct or nipple-shaped and solitary. Conidiophores (n= 30) were hyaline, erect, arising from the upper surface of hyphal mother cells or lateral, and of 90 to 201 µm long. Foot cells were cylindrical, of 49 to 92 × 10-15 µm, followed by 1-3 shorter cells, and forming conidia in chains. Conidia (n= 100) were hyaline, ellipsoid-ovoid, doliiform-subcylindrical, 27 to 40 × 14 to 20 µm. Conidial germination belonging to the Euoidium type. Chasmothecia were not observed. The morphological characters were consistent with those of Golovinomyces bolayi (Braun et al. 2019). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH451. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region was amplified by PCR using the primers ITS5/ITS4 (White et al. 1990) and sequenced. The resulting 506 bp sequence had 100% identity to those of G. bolayi (LC417109 and LC417106). Phylogenetic analyses using the Maximum Likelihood and Maximum Parsimony methods were performed and confirmed the results obtained in the morphological analysis. The isolate UACH451 grouped in a clade with isolates of G. bolayi. The ITS sequence was deposited in GenBank under accession number OR467546. Pathogenicity was confirmed by gently dusting conidia onto ten leaves of healthy lettuce plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 70%. All inoculated leaves developed similar symptoms to the original observation after 10 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. Based on morphological data and phylogenetic analysis, the fungus was identified as G. bolayi. This pathogen has been previously reported causing powdery mildew on lettuce in Argentina, Canada, Chile, Ecuador, Peru, USA and Venezuela (Braun et al. 2019; Mieslerová et al. 2020). To our knowledge, this is the first report of G. bolayi causing powdery mildew on lettuce in Mexico.

First report of the Horse nettle virus infecting tomato (Solanum lycopersicum L.) in the United States.

Tomato (Solanum lycopersicum L.), a member of the Solanaceae family, represents one of the most extensively cultivated vegetable species worldwide and traces its origin to western South America (Caruso et al. 2022). In a field survey conducted in 2023 in Bixby, Tulsa County, Oklahoma, distinct symptoms were noted in two plants: one exhibited mottling and cupping of leaves and brown discoloration on leaves, petioles, and stems, while the other displayed a downward curling of leaves. Leaf samples from both symptomatic tomato plants (labelled as K4 and K5) were collected, and total RNA was extracted individually via the TRI Reagent® method (Molecular Research Center Inc., Cincinnati, OH, USA). Subsequently, the RNA samples were pooled and subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, U.S.A.) at the genomic facility, Oklahoma State University (Stillwater, OK). Total read count of 8,227,020 (average length =150.5 bp) was obtained, trimmed, and de novo assembled using CLC Genomics Workbench v22.0.1 (QIAGEN) and used for BLASTn and BLASTx analysis. Two contigs: 6,375 bp (average coverage 2,915.92, read count 142,538) and 3,564 bp (average coverage 3,035.91, read count 82,370) from the pooled sample showed 88.6% and 96.7% nucleotide identities with RNA 1 (OP292294) and RNA 2 (OP292295) of Horse nettle virus A (HNA-A) isolate MD-1, respectively. Sequences of both partial contigs (RNA 1, accession no. PP063196) and RNA 2, accession no. PP063197) were submitted to GenBank. The HTS data did not reveal any other viral or viroid sequences in these two tomato samples. To further confirm the presence of HNV-A, total RNA from K4 and K5 samples was tested individually by RT-PCR using HNV specific primers (Supplementary Table 1) based on the two partial contig sequences. The expected PCR products (491 bp and 451 bp) were obtained only from the K4 sample and none from the K5 sample. PCR products were extracted from an agarose gel, cloned into the pGEM®-T Easy vector (Promega), and transformed into Escherichia coli DH5α cells (New England Bio Labs). Two clones for each PCR product were sequenced by Sanger sequencing. Nucleotide sequence comparisons and BLASTn analysis of 491 bp and 451 bp showed 86% and 97% nucleotide identity with RNA 1 and RNA 2 of HNV-A isolate MD-1 (OP292294 and OP292295), respectively. Additionally, eight more leaf samples from eight different symptomatic tomato plants were collected in the same field and tested by RT-PCR as described above. All eight samples were positive by RT-PCR, but no PCR band was obtained in the total RNA from a healthy tomato leaf used as a control. Sequences from the PCR products were identical to the obtained HTS sequences. Our results confirmed for the first time that HNV-A can infect tomatoes. Currently, HNV-A has been reported to only infect a single weed (Horse nettle, Solanum carolinense) (Zhou et al. 2023). The identification of HNV-A in tomatoes in Oklahoma suggests a potential host shift is of concern for local growers as well as tomato growers worldwide. This shift underscores the urgency for an in-depth investigation into the transmission and host specificity of HNV-A. This is the first report in the United States and the world that HNV-A could infect tomatoes naturally in a grower field.

First Report of powdery mildew of Quercus guyavifolia (Fagaceae) Caused by Erysiphe quercicola.

Oaks are the most abundant trees in naturally regenerated forests in China, play a crucial role in preventing soil erosion and maintaining ecological stability (Du et al. 2022). Quercus guyavifolia H. Léveillé (Fagaceae family, Subgenus Cerris, section Ilex), is endemic in China, distributed in the southeastern boundary of the Qinghai-Tibet Plateau, with elevations from 2, 000 - 4, 500 m a.s.l. (Denk et al. 2018; Sun et al. 2016). Powdery mildew is a prevalent disease of oaks with up to 60% of foliage infection, which can induce leaf necrosis or deformation and might contribute to oak decline (Marçais and Desprez-Loustau 2014). In September 2023, we found leaves of Q. guyavifolia near Yunnan Baima Snow Mountain covered with white fungal colonies. Diseased Q. guyavifolia plants were transplanted into a greenhouse at Yunnan University for pathogenicity tests. Conidia from diseased plants were blown into twenty healthy Q. guyavifolia seedlings by cold air blower and five non-inoculated healthy seedlings were used as control. The inoculated seedlings developed powdery mildew symptoms within ten days on both sides of the leaves. Trypan blue staining was used to identify the pathogen that infects Q. guyavifolia (Xiao et al. 2017). Microscopic examination revealed abundant conidia and extensive branched hyphae on leaves, similar to the characteristics of powdery mildew fungi. The mean length and width of conidia were 29.06 ± 3.96 × 9.52 ± 1.36 µm (n = 50). We collected fungi (YNBAIMAXS01) and extracted genomic DNA from five diseased plants (from the same location) using the CTAB method. We amplified and sequenced the ITS (Gardes and Bruns, 1993), MS294, and MS447 (two nuclear protein-encoding genes; Feau et al. 2011; GenBank numbers: PP079015, PP083693, PP083694). BLAST analysis revealed 100% identity of above three sequences with the ITS of Erysiphe quercicola isolate DACA010 (GenBank accession MT569439), MS294 of E. quercicola isolate GEM09_11_FRTB1 (GenBank accession KY348509), and MS447 of E. quercicola isolate A1I1.5 (GenBank accession KY466619). Therefore, the isolate YNBAIMAXS01 was identified as E. quercicola based on its morphological and molecular characteristics. Sequences from the above three regions for YNBAIMAXS01 and five Erysiphe species were used to construct a Maximum likelihood (ML) tree. In addition, we constructed a ML tree using only the ITS region of YNBAIMAXS01 and eight Erysiphe species from GenBank to better distinguish E. quercicola from these species. Both trees were constructed using MEGA X with K2 + G as best model. The ML trees confirmed the powdery mildew fungi isolated from Q. guyavifolia is closely related to E. alphitoides. To date, thirty-four powdery mildew species belonging to genus Erysiphe have been found affecting Quercus and nine oak species can be infected by E. quercicola (https://fungi.ars.usda.gov/). To our knowledge, this is the first report of powdery mildew caused by E. quercicola on Q. guyavifolia, thus the development of control strategies and disease management is urgently needed.

First Report of Fusarium pernambucanum Causing Fruit Rot of Melon in China.

In May of 2020, November of 2021 and May of 2022, a preharvest fruit rot with white mycelia was observed inside and outside of the fruits of thick skin muskmelon (Cucumis melo L.) growing in about ten greenhouses (each greenhouse had about 320 muskmelons) with disease incidence of 70% in Ningbo, Zhejiang Province of China. In order to identify the causal agent, plant tissues from the margin of the symptomatic tissue were sterilized for 1 min with 1% sodium hypochlorite (NaClO), 2 min with 75% ethyl alcohol, rinsed in sterile distilled water three times (Zhou et al 2019), and then placed on potato dextrose agar (PDA) plates containing streptomycin sulfate (100 µg/mL) at 25℃ for 4 days. Only Fusarium colonies were isolated from all the plant tissues. The growing hyphae were transferred to new PDA plates using the hyphal tip method, putative Fusarium colonies were purified by single-sporing. Six fungal isolates (Fi-1~6) were obtained. The average radial mycelial growth rate of Fusarium isolate Fi-3 was 4.6 mm/day at 25℃ in the dark on PDA, and like other five isolates. The colonies are abnormal, producing lots of aerial hyphae, each isolate was white to light orange. Isolate Fi-3 produced macroconidia with 4 to 6 septa, tapered with pronounced dorsiventral curvature and measured 21 to 30 µm long 4 to 5 µm wide on Spezieller Nährstoffarmer Agar (SNA) medium at 25℃ for 10 days (Leslie and Summerell 2006), but polyphialides and chlamydospores were still not available for 30 days. The pathogen species was further identified by translation elongation factor-1 alpha (EF-1α) sequencing. The EF-1α of six isolates were sequenced, and their EF-1α sequences were 100% identical to each other, and the sequence of strain Fi-3 was deposited in GenBank with accession no. OL782040 and was also compared with sequences in the FUSARIUM-ID database (Geiser et al. 2004), which indicated that it was 100% identical to those of F. pernambucanum strain NRRL 32864 (GenBank accession GQ505613), F. pernambucanum strain LC7040 (GenBank accession MK289626), and F. pernambucanum strain LC12149 (GenBank accession MK289588) within the Fusarium incarnatum - F. equiseti species complex 17 (FIESC17). Two phylogenetic trees were established based on the TEF1-α sequences of Fi-1~6 and other Fusarium spp., Fi-1~6 was clustered with the sequences of F. pernambucanum within the FIESC17. Thus, both morphological and molecular criteria supported identification of the strain as F. pernambucanum. A pathogenicity test was conducted to verify Koch's postulates, mycelium agar plugs (6 mm in diameter) were removed from the colony margin of a 3-day-old culture of strain Fi-3, healthy melon fruits were surface-sterilized with 70% ethanol and rinsed twice with sterile-distilled water. Then, the melons were wounded using a sterile inoculating needle to stab and inoculated by a mycelium agar plug of strain Fi-3 on the wound sites. 5 fruits were inoculated in each treatment, and a mycelium-free PDA plug was used as a negative control, repeated 3 times, at 25℃ with high relative humidity for 10 days. The results show disease symptoms similar to those naturally infected fruits on all inoculated melon fruits. The fungus re-isolated from the diseased fruits, showed the same colony morphology as the original isolate. Koch's postulates were repeated three times with the same results. Strain Fi-3 inoculated fruits without wounding remained healthy. To our knowledge, this is the first report of fruit rot of melon caused by F. pernambucanum in China.

Vascular streak dieback: A novel threat to redbud and other woody ornamental production in the United States.

Eastern redbud (Cercis canadensis L.) is a popular and high-value woody ornamental plant native to the eastern and south-central United States of America (U.S.A.). In recent years, redbud production in the Southeastern U.S.A. has been greatly affected by a novel threat: vascular streak dieback (VSD). Infected plants exhibit a common set of symptoms, including leaf scorch, tip dieback, and vascular streaking that creates a marbled pattern in stem cross-section. Based on both conventional diagnosis and molecular identification, it has been found that the fungus Ceratobasidium sp. D.P. Rogers (Csp) is consistently associated with VSD-symptomatic eastern redbuds. However, the causal agent(s) of VSD has not yet been conclusively confirmed. Although eastern redbud has been the most frequently identified host tree, more than 25 other native plant genera have been confirmed to have VSD associated with Csp. The near-obligate nature of this fungus has made it challenging to culture, extract DNA, and conduct further studies to confirm its pathogenicity. This article highlights the emerging challenges of VSD, focusing on the following: 1) the recent history of VSD; 2) the increasing importance of VSD to woody ornamental nursery production in the U.S.A.; 3) the currently available protocols for isolating, culturing, storing, and maintaining the putative causal agent; 4) the rapid molecular detection of Csp; 5) phylogenetic findings on the origin and relatedness of Csp to previously recorded diseases, especially VSD in cacao (Theobroma cacao L.); and 6) preliminary results and observations from fungicide trials and cultivar screening in Tennessee. The article also outlines research needed to comprehensively understand VSD and accelerate the development of effective management strategies.

First Report of Beet Western Yellows Virus Infecting Vasconcellea x heilbornii (Caricaceae) in Ecuador.

Vasconcellea x heilbornii, known as babaco, is a hybrid native to Ecuador grown in small orchards in sub-tropical highland regions. Over the last decade, several viruses have been identified in babaco using high-throughput sequencing (HTS) (Cornejo-Franco et al. 2020, (Reyes-Proaño et al. 2023). In 2021, total RNA from a babaco plant showing distinctive leaf yellowing was extracted using the PureLink RNA Mini Kit (Thermo Fischer Scientific, USA) and subjected to HTS on an Illumina NovaSeq6000 system as 150 paired-end reads (Macrogen Inc., South Korea). Library construction was done using the TruSeq Stranded Total RNA Sample kit with Plant Ribo-Zero, as described (Villamor et al. 2022). Reads were processed using BBDuk and de novo assembled using SPAdes 3.15. both implemented in Geneious 2022. Contig analysis was done by BLASTx using the NCBI viral sequence database (as of November 2022). HTS generated 54 million reads, of which 12% assembled into contigs corresponding to genomes of previously reported babaco viruses including babaco virus Q (BabVQ), babaco nucleorhabdovirus 1 (BabRV1) and babaco ilarvirus 1 (BabIV1). Interestingly, 144 reads (0.0003%) assembled into seven contigs ranging from 100 to 480 nucleotides (nt) in length. These contigs showed homology, with 97% amino acid (aa) identity (100% query coverage), to regions of the RNA-dependent-RNA-polymerase (RdRp) of beet western yellows virus (BWYV, Acc. No. NC_004756), a member of the Polerovirus genus. To confirm the occurrence of BWYV in babaco, double-stranded RNA (dsRNA) was extracted from 15 g of leaf tissue from the original sample as described (Dodds et al. 1984) and used as template for reverse-transcription (RT)-PCR using overlapping primers designed to span all short contigs. RT-PCR amplified fragments were cloned into a pGEM®T-easy vector (Promega, USA) and sequenced by the Sanger method (Macrogen Inc., South Korea). The sequences were assembled into a single 2.7 kbp BWYV genome fragment comprising the complete protein 1 (P1) and partial RdRp gene (GenBank Acc. No. PP480670). Sequence alignments between the partially sequenced genome of the babaco isolate and its corresponding fragment from the closest BWYV isolate (NC_004756) revealed 94% and 97% identities at the nt and aa levels, respectively. To assess the prevalence of BWYV in babaco, 30 leaf samples showing yellowing symptoms from Pichincha (n=15) and Azuay (n=15) provinces were tested by RT-PCR using total RNA. Total RNA extraction and reverse transcription were done using the methodology described by Halgren et al. (2007). For RT-PCR, the primer set BWYV_Bab_F: 5'-CAGTGTCCTCCAAGTGCAACAT-3' / BWYV_Bab_R: 5'GGTTCCTTCCCAGTTTGGTGGT-3', which amplifies a 235 nt-long P1 region, was used. Three RT-PCR products from each positive sample were purified using the GeneJET PCR clean-up kit (Thermo Scientific, USA) and sequenced. BWYV was confirmed in 9 out of 15 samples (60%) from Pichincha, and in 10 out of 15 samples (64%) from Azuay. Samples were also tested for additional babaco viruses as described (Reyes-Proaño et al. 2023). All BWYV-infected plants turned out positive for papaya ringspot virus (PRSV), babaco mosaic virus (BabMV), BabVQ, and BabIV1. Hence, the impact of BWYV infection on babaco plants in single and mixed infections warrants further investigation. To the best of our knowledge, this is the first report of BWYV in a crop in Ecuador, and the first time it has been found in a Caricaceae species.

First report of anthracnose caused by Colletotrichum fructicola on winter jasmine (Jasminum nudiflorum) in China.

Winter jasmine (Jasminum nudiflorum Lindl.) is a medium-sized, deciduous shrub native to China that has become a popular choice among gardeners and landscapers. In 2020 to 2021, symptoms of anthracnose including brown necrotic spots, enlarged irregular lesions and leaf blight were observed on leaves of 20 winter jasmine shrubs in a public garden (22°34'58'' N; 113°56'23'' E) in Shenzhen, China, and with an estimated disease incidence of 65%. Tissues samples (6 × 6 mm2) surrounding the necrotic spots were surface sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 1 min, then rinsed with sterile water for three times and dried with sterile filter paper. Tissues were placed on potato dextrose agar (PDA) medium and incubated at 25℃. After 3 to 7 d, pure cultures were obtained by transferring hyphal tips to new plates and 32 isolates producing Colletotrichum-like colonies were obtained from 40 tissues (isolation frequency=32/(4×10)=80%). Three representative isolates YCH09, YCH23 and YCH32 were selected for further study. Three selected isolates were identical in morphological characteristics. Colonies on PDA after 5 d at 25℃ were white to gray with cottony mycelia and grayish-white on the underside of the culture. Conidia (n = 60) measured 15.4 ± 1.1 µm (13.0 to 17.1 µm) in length and 5.4 ± 0.3 µm (4.9 to 6.0 µm) in width and were hyaline, single-celled, cylindrical with rounded ends. Appressoria (n = 15) measured 7.1 ± 0.1 µm (5.3 to 8.9 µm) in length and 5.2 ± 0.2 µm (4.1 to 6.2 µm) in width and were brown to dark brown, ovoid. These morphological features were aligned with those of Colletotrichum spp. (Weir et al. 2012). Sequences of five genetic markers of representative isolates YCH09, YCH23 and YCH32 including the rDNA internal transcribed spacer region, chitin synthase, partial actin, ß-tubulin 2 and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) region were 99.3 to 100% identical to the ex-type isolate of C. fructicola strain ICMP 18581 (Zhang et al., 2020). From the maximum likelihood phylogenetic tree which was constructed based on concatenated sequences, three representative isolates (YCH09, YCH23 and YCH32) were clustered with other isolates of C. fructicola. The above morphological and molecular characteristics suggest that causal agent was C. fructicola. Pathogenicity was tested using a whole-plant assay. Five healthy plants were inoculated by spraying a conidial suspension (1.5×104 conidia/ml; 20 ml per plant) of the isolate YCH23 onto the foliage (Marshall et al., 2023). Three noninoculated control plants were sprayed with sterile water. All plants were placed in a greenhouse at 25±2℃ with approximately 75% relative humidity. Yellow lesions appeared on leaves of inoculated plants as early as 4 days after inoculation (DAI), and irregularly shaped brown spots similar to those observed in the field were formed on 10 DAI. Noninoculated plants remained asymptomatic. Colletotrichum isolates resembling morphological characters of YCH23 were reisolated from all inoculated plants, then identified as C. fructicola by DNA sequence analysis. C. fructicola is a well-known fungus causing anthracnose on more than 63 plant species including agricultural and horticultural plants worldwide (Talhinhas and Baroncelli, 2021). To our knowledge, this is the first report of C. fructicola infecting J. nudiflorum plants in China. Since its potential risk to other horticultural plant species, precautions may be necessary to minimize the spread of this fungi.

First Report of Neofusicoccum yunnanense Causing Leaf Spot on Ivy in Yunnan Province, China.

Ivy (Hedera nepalensis var. sinensis (Tobl.) Rehd) is an evergreen root-climbing vine, widely cultivated in eastern Asia because of its ornamental, environmental, and medicinal value (Wu et al. 2019). In October 2023, the leaf spot symptom of Ivy was observed in the Kunming Botanical Garden in Yunnan Province, China (25.14°N, 102.75°E), and the disease incidence was up to 38% (76 of 200 leaves). Initially, dark-brown or black small spots appeared on the leaves. As the lesions progressed, their center emerged tawny, and the black halos were expanded around the lesions. In severe cases, small spots could link together to form leaf blight even resulting in blade death. In order to obtain pure isolates, 10 diseased leaves were collected and cut into 1 mm × 1 mm pieces, surface disinfected with 75 % ethanol for 30 s, followed by 3% NaClO for 3 min, and finally washed three times with sterilized water. The pieces were placed on the potato dextrose agar (PDA) media, which was incubated at 25°C for 3 days. Individual hyphal tips from the developing fungal colonies were placed on PDA and incubated for 5 to 10 days. Six strains (6 out of 10) were obtained with same colony morphology. Conidia were hyaline, unicellular, nonseptate, ellipsoidal or fusiform, thin walled, externally smooth and ranged from 15.0 to 22.0 (avg. 18.4) µm × 5.0 to 8.0 (avg. 7.2) µm (n=30). Morphological comparison proposed that the present fungi belonged to Neofusicoccum (Zhang et al. 2021). Two isolates (GUCC23-0141 and GUCC23-0142) were selected for multi-gene phylogenetic analyses. The PCR reaction of the internal transcribed spacer region (ITS), translation elongation factor 1-α (EF1-α), and ß-tubulin genes were run using primers ITS1/ ITS4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone and Kohn 1999), and Bt2a/ Bt2b (Glass and Donaldson 1995). The accession numbers of DNA sequences of GUCC23-0141 and GUCC23-0142 are ITS: PP728109 and PP728110; TUB2: PP744490 and PP744491; and TEF1-α: PP744488 and PP744489. BLAST analysis showed 100% identity for ITS and TUB2, and 98.97% for TEF1-α with the Neofusicoccum yunnanense (CSF6142). Phylogenetic analyses also supported that our isolates kept a close relationship to N. yunnanense. Three one-year plants were used for pathogenicity test, two of which were inoculated with PDA plugs containing N. yunnanense and one of which was inoculated with blank PDA plugs and used as control. For each plant, three leaves were selected to conduct the test, whose surfaces were sterilized with 75% alcohol. All the leaves were covered with cotton moistened with sterilized water on top. All plants were placed in a greenhouse with 25℃ and 75% humidity. Few small black spots were observed at the inoculation site after 3 days, which were enlarged gradually after 7 days. However, control plants remained healthy. N. yunnanense was reisolated from the diseased tissues and identified based on morphological and molecular characteristics. On basis of pathogen identification and Koch's test, we proposed the leaf spot of Ivy caused by N. yunnanense. This was the first report about N. yunnanense causing the disease of Ivy. This result provides a theoretical basis for further research into the control of the disease. As an important ornamental plant, we should pay attention to the management of this disease.

First report of Gnomoniopsis castaneae associated with branch dieback on chestnut tree (Castanea sativa) in Southern Chile.

European chestnut (Castanea sativa Mill.) currently reaches 1,470 ha, distributed from the Maule region to the Los Rios region in Chile. Almost 3000 tons of fruit have been exported in the last three years. A survey was carried out in January 2023 in an eight-year-old orchard located in Vilcún (38°34'46.22"S 72° 9'58.61"O), Araucanía Region. Chestnut trees with branch die back and reduced growth and vigor were detected. The incidence in the orchard was 3% (6 out of 200 trees) estimated by visual observation. Cross and longitudinal sections of the woody trunk of two trees were collected and examined, and an internal dark-brown discoloration to partial necrosis lesion was observed. To identify the causal agent, small pieces of wood from the edge of the symptomatic area were surface sterilized with 70% ethanol, rinsed twice with sterile distilled water, blotted on dry sterile filter paper, plated on potato dextrose agar (PDA) and incubated at 22°C. Fungal colonies were consistently isolated, and after 5 days, pure cultures were obtained by transferring mycelium to new PDA plates, preliminarily identified as Gnomoniopsis sp. (Visentin et al. 2012, Shuttleworth 2012). All cultures exhibited characteristics consistent with the description of G. castaneae (Syn. G. smithogilvyi), such as concentric development of greyish-brown mycelium, abundant stroma, hyaline conidia of 7.2 ±0.54 (6.1-8.1) X 2.3 ±0.26 (1.5-2.9) µm (n= 30), mainly biguttulate and fusoid. Total DNA was extracted, rDNA amplified using ITS1/ITS4 primers (White et al. 1990), and the fragment was Sanger sequenced and the sequence was deposited in GenBank (OR665735). BLAST analysis revealed a 99% identity to G. castaneae (MH384925). In addition, the DNA of the isolate was evaluated in a species-specific multiplex PCR (Silva-Campos et al. 2022), and the amplicons were electrophoretically separated, giving a similar band profile to G. smithogilvyi RGM 2903 and RGM 2904 strain from Chilean Collection of Microbial Genetic Resources. Pathogenicity of G. castaneae isolate (CV-11) was tested on ten replicates of 3-year-old C. sativa plants. Two wounds were made on the same season growing shoot and two on the previous season shoot. Longitudinal wounds (5 mm long, 4 mm wide and 2 mm depth) were made using a scalpel without removing the outer bark to inoculate the plants. Each wound was inoculated with a 5-mm mycelium plug, covered with the outer bark, and wrapped with Parafilm. Plugs of PDA were placed onto the wounds of two plants as control. The plants were kept in a growth chamber (22 ±1 0C and 90± 5% RH). All plants showed dark brown cankers measuring 20 to 40 mm long two weeks after inoculation. Also, most plants inoculated in the same season shoot presented wilted and chlorotic foliage. Mature conidiomata with cirri developed in most of the cankers. No symptoms were observed in the control. Fungal colonies of G. castaneae were reisolated on PDA from all inoculated chestnut plants and were not recovered from the controls. Recently, G. smithogilvyi has been identified as the causal agent of brown rot on chestnut nuts in Chile (Cisterna Oyarce et al. 2022); however, in several countries, it has also been associated as the causal agent of cankers in branch and stem of chestnut, as well as an endophyte in different hardwood species. Future studies on the incidence of this pathogen and its impact on chestnut yield should be carried out in the producing regions because it represents an emerging threat to Chilean chestnut production.

Ralstonia solanacearum species complex in Australia.

The Ralstonia solanacearum species complex (RSSC) causes vascular wilt of many crops and is considered one of the most destructive plant pathogenic bacteria worldwide. The species complex was recently resolved into a stable taxonomy of three species aligning with the previously determined phylotypes, namely R. solanacearum (phylotype II), R. pseudosolanacearum (phylotype I and III), and R. syzygii (phylotype IV). Knowing which Ralstonia species and subspecies are established in Australia is important to Australia's biosecurity and market access. The goal of this study was to analyse Australia's Ralstonia culture collections and to assign the isolates to the modern taxonomic groups. The results shed light on the identity, distribution, and pathogenicity of the Ralstonia strains in Australia. Ralstonia solanacearum, R. pseudosolanacearum phylotype I, and R. syzygii phylotype IV-11 are present in Australia but have limited geographic ranges. We identified two aberrant RSSC strains that have genetic similarity to R. syzygii based on sequevar analysis, but do not yield a phylotype IV multiplex PCR band, similar to the known aberrant strain ACH732. The aberrant strains may represent a novel species. Three new sequevars were determined, 72, 73 and 74. Several Ralstonia lineages remain undetected in Australia, providing evidence that they are absent. These include R. pseudosolanacearum phylotype III and the phylotype I mulberry infecting strains; R. solanacearum strains IIC and the Moko causing strains; and R. syzygii subsp. celebesensis, and R. syzygii subsp. syzygii. This study fulfilled Koch's postulates for the Australian strains, R. solanacearum wilted potato plants, and R. pseudosolanacearum wilted blueberry plants, the hosts from which they were initially isolated. The data supports the hypothesis that Australia has native and introduced strains of Ralstonia.

First Report of Mango Stem-end Rot Caused by Pestalotiopsis kenyana in Yunnan Province, China.

Stem End Rot (SER) is a devastating post-harvest disease of mango fruits causing severe losses during storage. In 22 July 2023, 31 out of 50 intact mangoes (cv. Sensation) collected from five orchards in Huaping county (26°37'N 101°15') showed typical symptoms of SER after stored for 9 d in room temperature (24-28℃). Initially, small dark brown to black spots appeared around the fruit peduncle, which rapidly expanded through the pulp tissues. The symptomatic mangoes were surface disinfected by 3% NaClO for 30 s after soaking in 75% alcohol for 3 min, and cleaned by sterile water for 3 times. Tissues were cut from the edge of lesions, dried by sterile filter paper, transferred to PDA and cultured at 28 ℃ for 5 d (Tovar-Pedraza et al., 2020). The single-spore isolation method was used to obtain pure culture. Thirty eight isolates presented four distinct kind of morphology on PDA medium. Among them, 11 isolates with same morphology were significantly distinct from common pathogens of SER. The colonies were white and pale yellow on reverse side. Mycelia grew fast and reached the edge of 90 mm Petri dish after cultured for 5d. Pycnidia were black and scattered on the mycelial mats after 15-20 d. Conidia were fusoid, straight to slightly curved, four septa, and brown. Pigmented median cells doliiform, 14.97 - 18.62(16.11 ±0.89)×5.61- 7.28 (6.61±0.51) µm. Apical cell hyaline, subcylindrical; 1-3 tubular transparent apical appendages 12.27 - 16.68 (13.65±3.78)×1.14 - 1.99 (1.59±0.36) µm. Basal cell conical with a truncate base, hyaline, and 1-2 tubulose basal appendages with 2.85 - 7.97 (5.18±1.88)×0.99 - 1.85 (1.38±0.29) µm (n=50). These fungi were described as Pestalotiopsis kenyana. based on morphological characters (Maharachchikumbura et al., 2014) which were different from isolates characterized as other common SER pathogens (Botryosphaeria, Neofusicoccum). Based on morphology, HPSX-4 was selected for further identification. ITS region, tef1-α, ß-tub of HPSX-4 were amplified and sequenced (Xun et al., 2023). The sequences were deposited in GenBank (ITS:OR889126, tef1-α:OR913431, ß-tub: OR913432). The ITS, tef1-α, ß-tub sequence of HPSX-4 showed 100% (525/525)，99.59% (241/242), and 100% (742/742) identity to the P. kenyana CBS442.67 sequences (ITS: NR147549，tef1-α: KM199502, ß-tub: KM199395), respectively. HPSX-4 clustered with P. kenyana CBS 442.67 (type strain) based on maximum likelihood method by MEGA 7.0.21(Minh et al., 2013). Pathogenicity test was performed on 12 healthy mangoes (cv. Golek) by placing mycelial plugs around the peduncle and the middle of the fruit by pin-prick method according to Feng et al.(2023). Sterile PDA were used as control (three mangoes). Every inoculated fruit was incubated at 28°C, 95% ± 3% humidity with three replicates for each treatment. The experiment was repeated three times. Typical symptoms of SER were observed. There were no symptoms in the control group. The strain was reisolated and identified as P. kenyana with the method mentioned above which fulfilled Koch's postulates. This is the first report of P. kenyana causing SER disease on Mangifera indica L.. This study expands our understanding of the pathogen range of mango SER which conducive to prevent and control the SER caused by P. kenyana.

First Report of Powdery mildew Caused by Golovinomyces ambrosiae on Verbena × hybrida in the U.S.

Verbena × hybrida, also known as common garden verbena, has an important ornamental value for their wide range of flower colors and for attracting hummingbirds and butterflies. During the winter of 2021-2022 (December through February), more than 50% pot-grown verbena plants showed symptoms of powdery mildew in a field trial at a Syngenta Crop Protection research facility in Vero Beach, FL. Symptoms were characterized by the development of white, superficial mycelium on the adaxial side of leaves which, eventually, progressed to covering the whole surface of leaves, causing leaf discoloration, shoot distortion, and eventual plant death. Morphological characterization was carried out by observing powdery mildew colonies under the microscope. This powdery mildew forms dense patches of white mycelia, mainly on the adaxial leaf surfaces. The mycelium was a mat of hyphae with septa. Conidiophores were erect. The foot cells were straight, followed by one to three short cells bearing short chains of up to four conidia. The conidia were hyaline and ellipsoidal to doliiform in shape. Conidial germination is of the Eudoidium type. The conidia ranged from 25 to 32 µm long by 12 to 16 µm wide. The length to width ratio ranged between 1.6 and 2.3, but most were between 2.0 and 2.2. This is further verification of its identity as Golovinomyces ambrosiae and not Golovinomyces latisporus, because the length to width ratio of the latter species is consistently less than 2.0 (Qiu et al. 2020). Chasmothecia were not observed. Additionally, the ITS, GAPDH, and IGS regions were sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), PMGAPDH1/PMGAPDH3R (Bradshaw et al. 2022a), and IGS-12a/NS1R (Carbone and Kohn 1999), respectively. The ITS region (GenBank number=PP924119) cannot distinguish between G. latisporus and G. ambrosiae and as such aligned 100% with both species on GenBank. However, the GAPDH and IGS regions can be used to distinguish G. ambrosiae from G. latisporus (Bradshaw et al. 2022b). The GAPDH (GenBank number=PP931995) and IGS (GenBank number=PP931996) regions aligned 100% with multiple G. ambrosiae sequences from GenBank including ON360708 and MK452567, respectively. The specimen was deposited in the Larry F. Grand Mycological Herbarium (NCSLG 24479). To confirm pathogenicity, 'Tuscany® Pink Picotee' and 'Quartz XP Violet with Eye' plugs were transplanted to 10-cm diameter pots containing ProMix potting mix and maintained in a greenhouse (± 26 °C). Inoculation was carried out 21 days after transplanting by touching infected leaves onto healthy leaves of 15 disease-free plants of each variety. Fifteen non-inoculated plants of each variety were used as controls. Typical powdery mildew symptoms and signs were first observed ten days after inoculation and the pathogen was more aggressive on 'Tuscany® Pink Picotee'. Symptoms were not observed on non-inoculated plants. The fungus was morphologically identical to the one originally recovered from infected plants in the field. There have been many reports of Golovinomyces spp. affecting Verbena spp. worldwide; however, this is the first report of G. ambrosiae causing powdery mildew on Verbena × hybrida in the U.S. (Braun and Cook, 2012, Choi et al., 2021; Bradshaw et al. 2024). Powdery mildews reduce plant quality and decreases the aesthetics value of infected plants, causing great losses to the ornamental industry. Correct identification of the causal agent is crucial to recommend appropriate control methods, as they may differ according to the pathogen species.

First report of basal rot of yellow dragon fruit (Selenicereus megalanthus) caused by Fusarium oxysporum in Peru.

Cultivation of yellow dragon fruit (Selenicereus megalanthus) in Peru has recently expanded (Verona-Ruiz et al. 2020). In August 2021, approximately 170 of 1,110 dragon fruit cuttings (15.3%) in the university's nursery (6°26'10'' S; 77°31'25'' W) showed basal rot symptoms. Initial symptoms included small brown spots on the base of stems, expanding towards the top that became soft and watery. All symptomatic plants eventually died, i.e., a severity of 100%. The disease was more prevalent on cuttings during the rooting phase than on well-established cuttings. We collected five symptomatic cuttings from throughout the nursery. Four sections of 1 × 1 cm2 of tissue adjacent to the diseased area were excised from each cutting, immersed for 1 min in 2% NaClO, rinsed twice with sterile distilled water, placed on potato dextrose agar (PDA) medium (four sections per Petri plate, five plates), and incubated at 25°C for 7 days. Morphologically similar mycelia grew from all sections, and five monosporic isolates were obtained, one per plate. Colonies grew fast, reaching 60 to 64 mm in 7 days, and produced violet-white cottony aerial mycelia with orange sporodochia on PDA, and abundant macro- and microconidia on synthetic nutrient-poor agar. Macroconidia were straight to slightly curved, typically with 2 to 3 septa, 16.6 to 23.3 × 1.7 to 3.7 µm (n = 30); microconidia were oval or kidney-shaped, and commonly hyaline, 6.7 to 16.4 × 2.5 to 4.7 µm (n = 40). Genomic DNA was extracted from isolate AFHP-100, then the ITS region and the TEF1 and RPB2 partial genes were amplified and sequenced (Accession numbers PP977433, OR437358, PP537149) following Gardes and Bruns (1993) and O'Donnell et al. (1998). We conducted a BLASTn search of ITS sequence against the NCBI "nr" database and local 'megablast' searches of TEF1 and RPB2 sequences against FUSARIUM-ID v.3.0 (Torres-Cruz et al. 2022). We found 100%, 98.19 to 99.84%, and 98.81 to 99.76% identities in ITS, TEF1, and RPB2 sequences, respectively, to the ex-epitype and other reference strains of Fusarium oxysporum (CBS 144134, NRRL26406, among others). A maximum likelihood phylogenetic analysis with a TEF1-RPB2 concatenated dataset with FUSARIUM-ID sequences also showed isolate AFHP-100 was F. oxysporum. A pathogenicity test was carried out by inoculating wounded healthy roots of three cuttings with submersion in a 5 × 106 conidia/ml suspension for 25 min. Then, the inoculated plants were planted in sterile soil. One cutting with wounded roots submerged in sterile water served as a control. In parallel, sterile soil was inoculated with 20 mL of the conidial suspension, and another three healthy cuttings were planted. A cutting planted in noninoculated soil also served as a control. Basal rot symptoms developed in all inoculated plants after 25 days. After re-isolation, the same fungus, corroborated based on micromorphology and TEF1 sequence (PP335689), was recovered, fulfilling Koch's postulates. The isolate was deposited in the KUELAP Herbarium (voucher KUELAP-3214), located and administered by the National University Toribio Rodriguez de Mendoza de Amazonas, in Chachapoyas, Peru. Fusarium oxysporum has been reported to cause basal stem rot in Bangladesh and Argentina (Mahmud et al. 2021; Wright et al. 2007), and stem blight in Malaysia (Mohd Hafifi et al. 2019) on dragon fruit. This is the first report of F. oxysporum causing basal rot in S. megalanthus in Peru. This fungus is among the most destructive plant pathogens, and the rapid expansion of the crop in Peru requires a comprehensive knowledge of the biotic factors influencing production. Therefore, this report is foundational to implementing proper control strategies.

First Report of Clonostachys rosea Causing Bulb Rot on Fritillaria taipaiensis in China.

Taibai Beimu (Fritillaria taipaiensis) is a species of Fritillaria commonly used in traditional Chinese medicine for its antitussive, expectorant, and antihypertensive properties. In April of 2021 and 2022, an incidence 10-30% of yellowing or purpling, wilting, and dying symptoms was observed on Taibai Beimu in Wanyuan, Sichuan province. Infected roots and bulbs displayed spots ranging from brown to black, along with necrotic rot. In severe cases, the entire bulbs rotted. Fifteen symptomatic bulbs were cut into 0.5 × 0.5 cm pieces, surface sterilized in 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min under aseptic conditions, rinsed with sterile water 3 times, and air-dried. The segments were placed on potato dextrose agar (PDA) and incubated at 25℃ for 7 days in the dark. Six Clonostachys-like monospore isolates were obtained. Colonies on PDA reached 32 to 43 mm in diameter in 7 days at 25℃ in the dark, felty to tomentose to granulose aerial mycelia with a white or light yellow appearance, and reverse colors matching. On cornmeal-dextrose agar, primary conidiophores had a Verticillium-like structure with 1 to 3 levels. Stipes were 36.1 to 236.3µm long. Phialides formed in whorls of 2 to 5, 15.3 to 45.7µm long, 1.1 to 3.4µm wide at the base, and 1.03 to 2.41µm wide near opening (n=95). Each producing a small hyaline drop of conidia. Conidia were 3.7 to 11.3µm × 2.1 to 4.1µm (n=110). Secondary conidiophores displayed Penicillium-like structures, and stipes were 23.1 to 142.3µm long. Phialides formed in compressed whorls of 4 to 8 per metula, 7.0 to 16.0µm in length, 1.3 to 3.1µm in width at the base, 1.8 to 3.6µm at the widest point, and 0.8 to 1.8µm near opening (n=50). Conidia were 3.0 to 6.4µm ×1.6 to 3.4µm (n=65). The morphology was consistent with the previous description of Clonostachys rosea (Hans-Josef et al. 1999). The ATP citrate lyase (ACL1), ß-tubulin (TUB2), translation elongation factor 1-α (tef1α), and the nuclear ribosomal internal transcribed spacer (ITS) of three strains were amplified and sequenced using primers acl1-230up/acl1-1220low (Gräfenhan et al. 2011), T1/CYLTUB1R (Crous et al. 2004; O'Donnell and Cigelnik 1997), EF1-728F/EF2 (Carbone and Kohn 1999; O'Donnell et al. 1998), and ITS1/ITS4 (White et al. 1990), respectively. Blastn homology search showed a > 97% similarity to the ex-type strains of C. rosea (CBS710.86). All sequences have been deposited in GenBank (PP394342 to PP394350, and PP396901 to PP396903). A phylogenetic tree was constructed using Bayesian analysis based on the alignment of the combined ACL1, TUB2, tef1α, and ITS sequences through IQ-TREE. The tree displayed clustering with known strains of C. rosea. Pathogenicity was confirmed by inoculating five healthy five-year-old Taibai beimu plants with a spore suspension (1.0 × 106 spores mL-1) of the strain WYEB1101, while sterilized water was used as a control. The inoculation process involved pouring the spore suspension over the wounded bulbs and covering with them sterile soil. Subsequently, all plants were cultivated in sterile soil indoors under natural conditions suitable for Taibai beimu. The pathogenicity assays were repeated twice. After 20 days of cultivation, the infected plants displayed symptoms similar to those observed in the field, while all control plants remained asymptomatic. Sequencing confirmed the re-isolation of C. rosea from the inoculated plants, satisfying Koch's hypothesis. Clonostachys rosea has been previously reported to cause root rot of Chinese medicine herb, such as Astragalus membranaceus and Gastrodia elata (Lee et al. 2020; Qi et al. 2022). To our knowledge, this is the first report of C. rosea infecting Taibai Beimu in China, highlighting a potential risk to this crop.