Cluster Zone Leaf Removal Reduces the Rate of Anthracnose (Elsinöe ampelina) Progress and Facilitates Its Control.

Anthracnose caused by Elsinöe ampelina is an economically important disease that affects certain hardy and semihardy grapevine cultivars. The control of this disease requires repeated application of fungicides, which has financial and environmental consequences. In this study, leaf removal in the cluster area was studied with a view to facilitating integrated anthracnose management. First, the effect of leaf removal timing (BBCH stage 53 or 71) and intensity (one or both sides of rows) on the progression of anthracnose and on the microclimate was studied in plots planted with Vidal blanc (Vitis vinifera) at two sites in both 2020 and 2021. Overall, at both sites and in both years, anthracnose on leaves was more severe in plots without cluster zone leaf removal. Regardless of the timing of leaf removal, anthracnose severity on leaves and incidence of infected berries at harvest were significantly lower in plots where leaves had been removed on both sides of the rows compared with plots where leaves were removed on one side only. Second, anthracnose management programs with leaf removal, with or without disease risk estimation, were evaluated. All anthracnose management programs including leaf removal in the cluster zone reduced anthracnose development compared with the standard program without leaf removal. Overall mean leaf anthracnose severity, severity at harvest, and anthracnose incidence on clusters at harvest were lower in plots with leaf removal than in the standard program, but the differences between the two treatments were not significant (P > 0.05). More fungicide applications were made in plots managed using the standard programs, specifically 13 applications, compared with plots managed based on assessing the weather-related risk of anthracnose, with 9 and 10 applications made at sites 1 and 2 for the risk-based program, respectively, and 5 and 7 applications made at sites 1 and 2, respectively, when microclimate within the cluster zone was considered. The results of this study clearly show the important role that leaf removal can play in managing grape anthracnose.

Detection and Characterization of Xylella fastidiosa subsp. fastidiosa in Rabbiteye Blueberry in South Carolina.

Xylella fastidiosa causes bacterial leaf scorch in southern highbush (Vaccinium corymbosum interspecific hybrids) and is also associated with a distinct disease phenotype in rabbiteye blueberry (V. virgatum) cultivars in the southeastern United States. Both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex have been reported to cause problems in southern highbush blueberry, but so far only X. fastidiosa subsp. multiplex has been reported in rabbiteye cultivars in Louisiana. In this study, we report detection of X. fastidiosa in rabbiteye blueberry plants in association with symptoms of foliar reddening and shoot dieback. High throughput sequencing of an X. fastidiosa-positive plant sample and comparative analyses identified the strain in one of these plants as being X. fastidiosa subsp. fastidiosa. We briefly discuss the implications of these findings, which may spur research into blueberry as a potential inoculum source that could enable spread to other susceptible fruit crops in South Carolina.

Quantitative Insights into Grapevine Anthracnose (Elsinoë ampelina) Epidemiology: Impact of Temperature and Leaf Age on Incubation, Lesion Development, and Sporulation.

Grapevine anthracnose, caused by Elsinoë ampelina, is one of the most devastating diseases for wine and table grapes, particularly in hot, humid regions. This study explores how temperature and leaf age affect incubation and how temperature affects lesion development and sporulation. The influence of temperature and leaf age on incubation period (days) was tested under controlled conditions. Leaves from 1 to 8 days old were inoculated and maintained at temperatures of 5, 10, 15, 20, 25, and 30°C. The time elapsed between inoculation and the emergence of initial lesions was recorded. The effect of temperature on lesion development and sporulation was investigated under vineyard conditions. This was achieved through artificial inoculations, with 17, 11, and 11 inoculations conducted in 2016, 2017, and 2018, respectively. The average incubation period, considering all leaf ages, was 27.50 days at 5°C, 15.10 days at 10°C, 9.70 days at 15°C, 5.90 days at 20°C, 3.70 days at 25°C, and 2.26 days at 30°C. Regardless of temperature, the average incubation period was 3.6, 5.9, 8.3, 9.8, 11.9, 13.4, 15.6, and 17.1 days for leaves 1, 2, 3, 4, 5, 6, 7, and 8 days old, respectively. The exponential decay model accurately describes the incubation period as a function of both temperature and leaf age. On average, the relative lesion development (RLD) was 0.00, 0.00, 0.23, 0.47, 0.72, 0.93, 0.92, 0.90, 0.94, and 1.0 at 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 days after inoculation, respectively. The average relative sporulation (RSPO) was 0.03, 0.36, 0.82, 0.96, and 1.0 at 5, 10, 15, 20, and 25 days after inoculation, respectively. Both RLD and RSPO as a function of degree-days (Tbase = 0°C) since inoculation were well described by the logistic function. The rates of change in RLD and RSPO were 0.055 and 0.032, respectively. The results of this study provide new quantitative insights into three important stages (monocyclic processes) in the development of grapevine anthracnose caused by E. ampelina.

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 Neofabraea actinidiae causing a Cranberry Fruit Rot in Oregon.

Cranberry (Vaccinium macrocarpon, L.) is a commercial small fruit that is native to North America. Oregon ranks fourth in cranberry production in the U.S.A. with 1052 Ha of cranberry beds and annual production of 23,590 metric tonnes (USDA NASS 2021). Cranberry fruit rot diseases are caused by a complex of 15 fungal pathogens belonging to 10 genera (Polashock et al. 2017). In fruit rot surveys of 'Stevens' cranberry beds in Coos and Curry Counties, Oregon, berries were collected before harvest and sorted by symptoms (e.g. softening, shriveling, or discoloration). Cranberries with field rot symptoms were surface-disinfested and lesion margin tissue placed on V8 agar. Cultures were incubated at room temperature and outgrowing fungi were transferred twice onto fresh V8 agar to obtain single isolates. Storage rots that developed on asymptomatic cranberries held at 4°C for 8 weeks were processed similarly. Since 2017, we periodically isolated Neofabraea actinidiae, which is not a member of the cranberry fruit rot complex, from rotted cranberries (Polashock et al. 2017). In 2022, N. actinidiae was isolated from 22% of 45 cranberries collected from an organically managed farm and developed storage rot and from 6% of 50 storage-rot cranberries from three conventionally managed farms. Symptoms associated with N. actinidiae on 'Stevens' cranberries often include softening and brown tissue surrounded by a yellow-colored ring. On V8 agar, N. actinidiae grew as compact white circular colonies with dense aerial hyphae near the center and accompanied by a red pigment in the agar. Pink-colored mucoidal irregular conidiomata often developed on the colony after 3 weeks. Conidia were hyaline, aseptate, and ellipsoidal to fusiform ranging from 7.5 to 12.6 µm long X 3.5 to 5.6 µm wide (n=100). Genomic DNA was extracted from N. actinidiae isolates from cranberries in 2017 and 2022. ß-tubulin and the ITS 5/4 region were amplified and sequenced with primers Bt-T2m-Up/Bt-LEV-Lo1 and ITS5/ITS4 using conditions of de Jong et al. (2001) and White et al. (1990), respectively. Sequences were deposited in NCBI Genbank (Accessions: OR606303, OR606305, OR606306, & OR606309 for ITS; OR610314, OR610316, OR610317, & OR610320 for ß-tubulin). BlastN analysis of ß-tubulin (695 bp) and ITS (489-490 bp) had a 99.6 to 99.8% and 99.8 to 100% identity, respectively, to Neofabraea actinidiae (CBS 121403) (Accession numbers: KR859285 ß-tubulin and KR859079 ITS). Phylogenetic analysis of concatenated sequences using Tamura-Nei neighbor-joining (Tamura et al. 2004) confirmed identity of cranberry isolates as N. actinidiae. Koch's Postulates were confirmed with four N. actinidiae isolates from cranberry. Agar or hyphal plugs were placed in a 3 mm wound on the side of six surface-disinfested, asymptomatic berries and incubated in a moist chamber at 20°C for 15 days or 4°C for 55 days. Similar symptoms developed on each berry inoculated with N. actinidiae, but not agar alone. The fungus was recovered from symptomatic tissues and identity confirmed by colony morphology. N. actinidiae causes a ripe rot and storage rot in kiwifruit and is one of the species causing Bull's Eye Rot of pome fruits (Tyson et al. 2019). Cryptosporiopsis actinidiae (anamorph) was isolated from cranberries roots in British Columbia, CA, but considered unlikely to be the causal agent of dieback disease of cranberry vines (Sabaratnam et al. 2009). We have demonstrated that N. actinidiae causes a cranberry fruit rot in beds and in storage. Its prevalence, associated fruit rot symptoms, and disease incidence will continue to be monitored.

First Report of Lingonberry Stunted Yellows Disease of Vaccinium vitis-idaea L. associated with 'Candidatus Phytoplasma trifolii'-Related Phytoplasma Strain in Lithuania.

Lingonberries (Vaccinium vitis-idaea L.) are low-growing, evergreen shrubs of cooler, northern regions of North America and Europe. These plants produce berries that are unique in flavor, bear high economic significance, and play a vital role in maintaining the diversity of the northern ecosystems (Kowalska, 2021). In October 2023 diseased plants of lingonberry were discovered in Labanoras Forest (55°14'N 25°42'E) (Lithuania). The plants expressed symptoms of stunting, yellowing, little leaf, shortened internodes, and stem distortions. Samples (leaves) were collected and tested from ten asymptomatic and ten symptomatic lingonberry plants. Total genomic DNAs of all samples were extracted by a CTAB protocol. Extracted DNAs were used as a template in direct and nested PCRs using the universal primer pairs P1/P7 and R16F2n/R2, respectively, to amplify phytoplasma 16S rRNA gene 1.2 kb fragments (Lee et al. 1998). The primer pairs SecAFor1/SecARev3 and SecAFor2/SecARev3 were used in direct and semi-nested PCRs, respectively, to amplify phytoplasma secA genes 0.5 kb fragment (Dickinson and Hodgetts, 2013). PCR amplicons of the 16S rRNA and secA genes specific for the phytoplasmas were only obtained from all sampled symptomatic plants. Three R16F2n/R2 and three SecAFor2/SecARev3 amplicons were cloned and submitted for Sanger sequencing (Nature Research Centre, Vilnius, Lithuania by 3500 Genetic Analyser). The three 16S rDNAs as well as the three secA gene fragments were identical. The BLAST analysis (NCBI) of the obtained sequences showed a similarity percentage, ranging from 99.75% to 100% (1247-1250 bp from 1250 bp) for 16SrRNA, and 98.13% to 99,15% (473-478 bp from 482 bp) for secA amplicons, with numerous strains of 'Candidatus (Ca.) Phytoplasma (P.) trifolii' (first hit MT674293 and KR906724, respectively). Additionally, 16S rDNA sequences by using iPhyClassifier were used to create virtual RFLP pattern (Zhao et al. 2009). The generated pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A. The phytoplasma strain detected in lingonberries was designated as lingonberry stunted yellows, LingbSY. Furthermore, the enzymatic RFLP analysis was performed with the 14 restriction enzymes (Lee et al., 1998), and obtained profiles were compared with virtually generated using iPhyClassifier. This yielded the same classification of detected phytoplasma to the 16SrVI-A phytoplasma subgroup. The phylogenetic analysis of both marker gene sequences revealed the same LingbSY phytoplasma classification. Selected sequences were deposited in GenBank (NCBI) with Accession No: PP237769 (16S rRNA gene) and No: PP238489 (secA gene). Phytoplasmas of 16SrI phytoplasma group were identified in lingonberries in Canada (Brochu et al. 2022). Strains of 16SrVI phytoplasma group were reported in Vaccinium myrtillus in Austria (Fernandez et al. 2007). This is the first report of 'Ca. P. trifolii' strain belonging to 16SrVI-A phytoplasma subgroup infecting lingonberry worldwide. Also, this is the first report of 16SrVI phytoplasma group in Lithuania. The presence of this phytoplasma poses a threat to the natural ecosystem and could eventually spread into agricultural settings in our country. Therefore, it's crucial to conduct surveillance for insect vectors, and assess effective control methods. Without proactive action, long term sustainability of lingonberries and their ecosystems may be jeopardized.

First report of root rot of strawberry caused by Fusarium cugenangense, a member of the F. oxysporum species complex, in Tennessee, USA.

Strawberry (Fragaria × ananassa Duch) in Tennessee is cultivated on plastic mulched beds annually, and production is limited primarily by multiple oomycete and fungal root rot pathogens that result in reduced vigor and black root rot disease symptoms. In early June 2018, plants (cv. Chandler) with reduced shoot vigor and size, and black, necrotic stunted roots were collected from Rhea County, TN. Roots and crowns of 10 plants were cut into 1-3 cm pieces and surface sterilized with 0.6% NaOCl, followed by 70% ethanol for 1 min each, and plated on water agar. White mycelia produced after 3 days were transferred to potato dextrose agar amended with 10 mg/liter rifampicin. After 10 days, fungal colonies were light purple on the surface and dark purple on the colony underside, later developing blue-black pigmentation on the underside. Microconidia on carnation leaf agar were ovoid to ellipsoid, aseptate or septate and 8.0 to 24.2 (13.7) × 3.0 to 4.5 (3.8) µm in size, macroconidia were 3 to 5 septate and falcate to almost straight and 33.7 to 52.8 (44.4) × 4.0 to 5.5 (4.9) µm in size (n=80); both conidia were produced on monophialides. Chlamydospores were globose and subglobose, formed terminally and intercalary on aerial, submerged, and surface mycelium, singly or in pairs and were abundantly produced in sucrose broth and on synthetic nutrient-poor agar (SNA) (diam. 7.6 µm). Morphology was consistent with Fusarium oxysporum (Leslie and Summerell, 2006) and F. cugenangense, a member of the F. oxysporum species complex, as described by Maryani et al. (2019). Fungal mycelia were used for PCR (Phire Plant Direct PCR Master Mix, Thermo Scientific, CA) and the translational elongation factor 1-α (EF1α) region was amplified with primers EF-1/EF-2 (O'Donnell et al., 1998), internal transcribed spacer (ITS) regions amplified with primers ITS1/ITS2 (White et al. 1990), and the RNA polymerase second largest subunit region (RPB2) with primer pairs 5f2/7cr and 7cf/11ar (O'Donnell et al., 2022). PCR products of isolate SC5 were sequenced, and sequences compared to all sequences in the FUSARIOID-ID database using polyphasic identification (Crous et al., 2021) with EF1α (GenBank Accession No. ON703236) and RPB2 (OR472390) sequences. The highest similarity (100%) was with isolates of F. cugenangense, including ex-type isolate InaCC F984 (99.94% similarity) (Maryani et al., 2019). F. cugenangense is closely related to F. callistephi and F. elaeidis, but both species lack chlamydospores, and F. elaeidis has polyphialides (Lombard et al, 2019). To satisfy Koch's postulates, healthy rooted strawberry plants produced in soilless media were transplanted into 4 plastic pots (1.2-liter) containing 5% (w/v) fungal inoculum (grown on barley grain) and mixed into the top 5-cm of peat-based soilless medium. Pots were incubated at 25°C and 50% RH in a growth chamber. Four pots without inoculum served as controls. The trial was repeated. Within 8 weeks, all inoculated plants had low vigor, with necrotic and stunted roots. Root sections of control and inoculated plants were plated, and the pathogen was re-isolated from diseased roots of all inoculated plants only and confirmed as F. cugenangense based on morphology and sequence analysis. To our knowledge, this is the first report of F. cugenangense, or any member of the F. oxysporum species complex, causing root rot of strawberry in Tennessee and could be an important component of the production-limiting black root rot disease complex of strawberry.

Characterization and fungicide sensitivity of Gnomoniopsis fructicola causing Gnomonia Leaf Blotch of strawberry in the Carolinas.

Emerging fungal pathogens have always been an issue of concern in southeastern U.S. strawberry production. In 2023, an unusual outbreak of Gnomonia leaf blotch occurred at one North Carolina (NC) and multiple South Carolina (SC) strawberry farms and marked the first report of its occurrence in SC. Molecular identification and phylogenetic analysis of isolates from multiple locations identified the fungus Gnomoniopsis fructicola as the causal agent. In vitro germination of G. fructicola progressed slowly and remained less than 40% even after 24 h of incubation. Similarly, germ tube growth was slow compared to other pathogens. Slow symptom development on strawberry leaves of young strawberry plants grown in the greenhouse started 5 weeks after inoculation. Once the pathogen established on greenhouse plants, leaf necrosis forming blotches was observed. The baseline sensitivity of G. fructicola isolates to commonly used chemical classes of fungicides was assessed. Propiconazole, cyprodinil, pyraclostrobin, and fludioxonil were highly effective in mycelial growth assays with EC50 values < 0.01 µg/ml. Iprodione and thiophanate-methyl were also effective with EC50 values ranging from 0.05 to 1.38 and 2.01 to 23.96 µg/ml, respectively. Fluopyram and fenhexamid were ineffective with EC50 values >100 µg/ml. Based on conversations with the producers, the disease outbreak was linked to transplants from the same nursery source. This study reports for the first time the presence of Gnomonia leaf blotch in South Carolina and provides valuable insights into chemical management options for G. fructicola.

First report of the new Neopestalotiopsis species causing strawberry leaf spot and fruit rot in Georgia.

In April 2023, symptomatic strawberry (Fragaria × ananassa) plants (cv. 'Camarosa' and 'Florida Brilliance') were observed at a commercial farm in Worth County, GA (USA). Symptoms included foliar, irregularly distributed, and different-sized spots (dark brown with light brown centers) and dark brown V-shaped necrotic areas starting at the leaf edge. By the time of sample collection, ~50% incidence was reported in the field. Leaf samples were collected and shipped overnight to the laboratory. Black acervuli were observed readily on old necrotic foliar lesions. Conidial morphology was consistent with that observed with Neopestalotiopsis species (Maharachchikumbura et al. 2014). Conidia were ellipsoid to fusiform, five-celled, with three light brown median cells and one hyaline apical and basal cell. Apical cells had two-to-four flexuous appendages, and the basal cell had one non-flexuous appendage (Fig 1). The average (n = 20) conidia length, not including the appendages, was 26.6 µm (SD: 2.8), and width was 6.3 µm (SD: 0.94). Fungal isolation was conducted on acidified PDA and incubated at 25°C for 6 days. Dense, white mycelia were observed on the upper plate surface, while a pale pink/orange coloration was observed on the underside (Fig 1). Black acervuli formed on the surface of the white mycelial mat. Six isolates were purified and selected to confirm the species identity. DNA was extracted from 6-day-old cultures and PCR was conducted following Kaur et al. (2023). Amplified DNA was digested with the restriction enzyme BsaWI and two bands were clearly visualized (~130 and ~290 bp), along with a faint band of 20-bp (Fig 2). Four of the six isolates were selected for sequencing of the ß-tubulin gene. BLAST queries using the consensus sequence showed that all isolates had 100% identity to strain N21002 from Florida (FL), characterized as Neopestalotiopsis sp. (Kaur et al. 2023). One representative isolate (AJ07-2023) was deposited in GenBank (accession No. PP316103). Pathogenicity tests were performed on 27-day-old transplants of Sensation 'Florida127' provided by Natalia Peres from the UF. Plants were grown in 10.5 cm pots in the greenhouse. Isolate AJ07-2023 was grown on PDA for 30 days at 25°C, and the spore suspension was adjusted at 106 spore/ml. Five strawberry plants were sprayed with 5 ml of inoculum using a Preval sprayer with a CO2 canister, and 5 plants were sprayed with sterile distilled water. Plants were placed in a growth chamber for 6 days and covered with plastic bags after the sixth day to maintain ~85% relative humidity and 25°C. Foliar symptoms, including dark-brown circular lesions occurring towards the edge of leaves with light-brown center and light-yellow halo, developed 13 days after inoculation. No symptoms were observed on control plants. Neopestalotiopsis sp. was reisolated from inoculated plants as described above. Colony, conidial morphology, and PCR results were consistent with the original isolates. Neopestalotiopsis disease has been reported on strawberry in FL (Baggio et al. 2021), OH (Rotondo et al. 2022) and IN (Guan et al. 2023). Although the disease has been observed sporadically in GA since 2020 (Brannen, personal communication), to our knowledge, this is the first official report of the new Neopestalotiopsis sp. in GA. It has been reported that this new strain is more aggressive on fruits and leaves than other Neopestalotiopsis spp. (Baggio et al. 2021), therefore, accurate identification is critical for proper management.

First Report on the Occurrence of Tomato Yellow Leaf Curl Virus in Tennessee.

In the summer of 2021, a field survey of several tomato-growing counties in Tennessee (TN) was conducted for plants exhibiting virus-like symptoms. While scouting in September in Grainger County, one of the largest areas under tomato (Solanum lycopersicum) production in TN, leaves from six tomato plants (cv. BHN 589) growing on a farm located near Rutledge were collected and subsequently stored at -80˚C. Only one of the plants exhibited symptoms typical of tomato yellow leaf curl virus (TYLCV) infection, which included chlorosis, leaf curling, downward cupping, thickening, and mottling. Total DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Santa Clara, CA) and subjected to PCR using primers TYv2337F (5'-ACGTAGGTCTTGACATCTGTTGAGCTC-3') and TYc138-R: (5'-AAGTGGGTCCCACAATTGCAAGAC-3') and Ex-Taq polymerase (Takara Bio, Mountain View, CA) to amplify a 634-bp genomic fragment of TYLCV (Alkowni et al. 2019). Primers against tomato elongation factor-1 served as internal PCR control (Dias et al. 2023). Each primer set amplified amplicons of expected sizes; however, the TYLCV fragment was detected only from the plant exhibiting typical symptoms of infection. Amplicons were purified with the QIAquick PCR purification kit (Qiagen) and sequenced directly bi-directionally by Eurofins USA using the above primers. The resultant sequences were edited and analyzed with CLC Genomic Workbench v. 24.0.1. Blast analysis of the sequences (606 nts) against those available in GenBank showed 93 TYLCV isolates with over 95% nucleotide sequence identity. Subsequently, the full-length genome was PCR amplified using primers TYBamHIv (5'- GGATCCACTTCTAAATGAATTTCCTG-3') and TYBamHI2c (5'-GGATCCCACATAGTGCAAGACAAAC-3') (Rojas et al. 2007), ligated into pGEM-T (Promega, Madison, WI) and cloned. Plasmids were purified using QIAprep Spin Miniprep kit (Qiagen) and five independent plasmids clones were sequenced using Oxford Nanopore sequencing (v14 library chemistry & R10.4.1 flow cell) by Eurofins USA. The resultant sequences were edited and analyzed with CLC Genomic Workbench and a consensus sequence representing the full-length genome (2,781 nts) was generated and submitted to GenBank (Accession No. PP505780). Blast analysis showed over 98% nucleotide sequence identity with 100 TYLCV isolates from GenBank. The highest sequence identity of 98.6% was with the sequence of an isolate from Florida (AY530931). To the best of our knowledge, this is the first report of the occurrence of TYLCV in TN. The virus was detected in a tomato plant grown from seed. The seed transmissibility of TYLCV remains controversial (Perry 2018; and references therein); thus, the most likely source of infection in this report is transmission by rare viruliferous vectors (Bemisia tabaci). It remains unknown, however, whether TYLCV is endemic in TN, or recently introduced by mobile vectors from neighboring states. The presence of TYLCV has been reported in Alabama (Akad et al. 2007), Kentucky (de Sá et al. 2008), Mississippi (Ingram and Henn 2001), Georgia (Momol et al. 1999) and North Carolina (Polston et al. 2002). The B. tabaci vector of the virus has sporadic occurrences in crops within TN (Li et al. 2021). Tennessee is one of the leading tomato producers exporting globally with production covering over 1,300 hectares and over 430 producers (Dias et al. 2023). Because of the potential threat of TYLCV to tomato industry in the state, additional surveillance measures need to be put in place to determine TYLCV incidence.

First Report of Leaf Spot on Cucumis melo L. Caused by Arcopilus aureus in China.

Cucumis melo L. is an important fruit with widespread consumption and commercial value. However, an undescribed disease affecting Hami melon (Cucumis melo L. var. Luhoutian) plants has consistently emerged in the Qihe region of Dezhou, Shandong Province of China since 2021. The disease can occur in both seedling and mature stages of Hami melon plants, and in severely diseased areas, the incidence rate was seen as 40 to 80%. During the seedling stage, the initial symptom is the appearance of water-soaked spots on the leaves. As the disease progresses, the leaves develop necrotic spots, and severely affected plants may exhibit stem rot and decay. In the mature stage, the disease primarily affects the leaves, causing necrotic spots and chlorosis. Under conditions of high humidity, black mold can be observed in the affected areas. Small pieces of symptomatic leaves from six different infected plants were collected and surface-sterilized with 5% NaClO for 3 min and 75% alcohol for 30 s for pathogen isolation (Wang et al., 2020). After rinsing with sterile water and blotted on sterile filter paper, the tissues were established on potato dextrose agar (PDA) media and incubated at 28℃ for 3-4 days. Pure isolates showed up at PDA were obtained through single-spore isolation. Colonies of all 16 isolates obtained by single-spore isolation had similar morphological characteristics on the PDA medium, the mycelium of the isolate appears dense and yellowish-brown on the PDA medium, and also secretes a brownish-red pigment on PDA. Under the opticalmicroscope, the perithecia from PDA media are subglobose spherical in shape, 80-100 µm in diameter, brownish by reflected light, wholly and densely hairy. Terminal hairs are very dense, greyish by reflected light, olive brown to reddish brown by transmitted light, thick-walled, arcuate, circinate, or spirally coiled at the apex. The ascospores within the perithecia are elliptical or droplet-shaped, initially colorless hyaline but later becoming subhyaline slightly gray, with dimensions of 7-9 µm × 4-5 µm. The morphological characteristics of the isolates were consistent with the description of Arcopilus aureus (Wang et.al. 2016). The internal transcribed spacer (ITS) region and ß-tubulin genes of three randomly selected isolates were PCR amplified and sequenced using primers ITS4/ITS5 and Bt2a/Bt2b. The sequences of ITS and ß-tubulin genes were submitted to NCBI with GenBank Accession No. OR539527 and OR640972, respectively. Based on morphological features and phylogenetic analysis, we concluded that the isolates belonged to A. aureus. Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of Hami melon seedlings (n=12) grown at 28°C with 60% humidity in a greenhouse, the assay was repeated three times. Symptoms appeared on the pathogen-inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as A. aureus based on morphological, and molecular phylogenetic analysis, while Koch'sostulate was used to confirm its life mode. To the best of our knowledge, this is the first report of leaf spot caused by A. aureus on Cucumis melo L. in China.

First report of leaf spot disease caused by Pseudopithomyces chartarum on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) cultivation has gradually expanded in China but continues to be limited by challenges such as leaf spot disease. Between September 2022 and September 2023, a leaf spot disease was observed on approximately 30% of 'Lanjingling' blue honeysuckles grown in a 2.66 ha field (a total of about 11,000 plants) in Jiamusi city (130.47°E, 46.16°N), Heilongjiang Province, China. Affected plants displayed brown necrotic lesions on their leaves that gradually expanded in area until the leaves fell off the plant entirely. Small, 3 to 4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Yan et al. 2022). Five pathogens (LD-232, LD-233, LD-234, LD-235, and LD-236) were isolated on PDA and displayed a conidia morphology consistent with Pseudopithomyces spp. (Perelló et al. 2017). The fungal colonies on PDA were villiform, white, and whorled and had sparse aerial mycelium on the surface with black conidiomata. The conidia were obpyriform and dark brown, had 0 to 3 transverse and 0 to 1 longitudinal septa, and measured 9.00 to 15.30 µm × 5.70 to 9.30 µm in size (n = 50). Genomic DNA was extracted from a representative isolate, LD-232, for molecular verification and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), LROR/LR7 (Carbone and Kohn 1999), and RPB2-5F2/RPB2-7CR (Liu et al. 1999) primers. Sequences of LD-232 ITS (OR835654), LSU (OR835652), and RPB2 (OR859769) revealed 99.8% (530/531 nt), 98.8% (639/647 nt), and 99.8% (1015/1017 nt) shared identity with Pseudopithomyces chartarum sequences (OP269600, OP237014, and MK434892), respectively (Wu et al. 2023). Bayesian inference (BI) was used to construct the phylogenies using Mr. Bayes v. 3.2.7 to confirm the identity of the isolates (Ariyawansa et al. 2015). Phylogenetic trees cannot be constructed based on the genes' concatenated sequences because selective strains do not have complete rDNA-ITS, LSU, and RPB2 sequences. Therefore, based on the morphological characteristics and molecular phylogeny, LD-232 was identified as P. chartarum (Perelló et al. 2017; Wu et al. 2023). A pathogenicity test was performed with six healthy, two-year-old 'Lanjingling' blue honeysuckle plants. Three plants were inoculated by spraying the LD-232 conidial suspension (1 × 106 spores/ml) or clean water as an experimental control condition (Wu et al. 2023; Yan et al. 2023). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was replicated three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 days. The same pathogens were reisolated from infected leaves, displayed the same morphological and molecular traits, and were again identified as P. chartarum, confirming Koch's postulate. P. chartarum previously caused leaf spot disease on Tetrapanax papyrifer in China (Wu et al. 2023). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by P. chartarum in China. Identification of P. chartarum as a disease agent on blue honeysuckle will help guide future management of leaf diseases for this economically important small fruit tree.

First Report of Anthracnose Caused by Colletotrichum plurivorum on Passion fruit in Guangxi, China.

Passion fruit (Passiflora edulis Sims.) is popular for its rich taste and nutritional value. The planting area of passion fruit in Guangxi has reached 24,300 ha, with an annual output of 380,000 t (Qian 2023). In March 2023, leave spots on more than half of the plants (cv. Qinmi "NO.9"). Moreover, the incidence of disease on the leaves was approximately 20% in Shabu Town, Qinnan District, Qinzhou City, Guangxi, China (N20˚54'-22˚41', E107˚27'-109˚56'). Leaf diseases were orbicular or irregular in shape, white, whitish-grey, yellowish, or gray in color. When leaves were severely affected, larger blotches were formed with yellow halos. For pathogen isolation, three diseased leaf samples were collected from three gardens, respectively, and 5×5 mm tissues were cut from infected margins, surface-disinfected in 75% ethanol for 15 s, followed by 2% sodium hypochlorite for 1 min, rinsed three times with sterile water, and incubated on PDA at 25°C under 12/12 h light/darkness. After 5 days, ninety cultures were isolated, sixty isolates with similar morphology were retained, and three representative isolates BY-1, BY-2, and BY-4 were randomly selected for further study. On PDA, colonies of the three isolates displayed white or grayish-white. Conidia were single-celled, hyaline, and cylindrical, measuring 17.3±1.5 × 6.3±0.7 µm, 17.8±1.7 × 6.0±0.6 µm, and 16.3±1.4 × 6.4±0.6 µm (n=90) for BY-1, BY-2, and BY-4, respectively. Appressoria were single, brown or black, and irregular in shape, measuring 10.2±1.1×6.5±0.5 µm, 10.5±1.3×7.3±0.6, and 10.9±0.8×7.0±0.8 (n=90) for BY-1, BY-2, and BY-4, respectively. These morphological characteristics were similar to Colletotrichum spp. as previously described (Damm et al. 2019). The isolates were further identified by sequencing the internal transcribed spacer (ITS-ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH-GDF/GDR), actin (ACT-512F/783R), partial sequences of the chitin synthase 1 (CHS-1-79F/354R), and beta-tubulin 2 (TUB2-T1/Bt2b) (Zhang et al. 2023). All sequences were deposited in GenBank (ITS: OR741759 to OR741761, GAPDH: OR767654 to OR767656, ACT: OR767657 to OR767659, CHS-1: OR767660 to OR767662, TUB2: OR767651 to OR767653). A phylogenetic tree was built with RAxML version 8.2.10 based on concatenated sequences of ITS-GAPDH-ACT-CHS-1-TUB2. The results revealed that the three isolates clustered with C. plurivorum. To confirm the pathogenicity of the three isolates, attached leaves of healthy 5-month-old passion fruit plants were injured in the middle region with sterile toothpicks and inoculated with 20 µL of spore suspension (106 conidia/mL), and the noninoculated control received 0.05% Tween-20 (6 leaves/plant, 3 plants/treatment). The inoculated plants were kept in a greenhouse at 25°C and covered with plastic bags to maintain high humidity. After 9 days, all inoculated leaves were symptomatic, whereas no symptoms were observed in the control. C. plurivorum was reisolated from infected leaves, confirming Koch's postulates. C. plurivorum has been reported to infect Abelmoschus esculentus (Batista et al. 2020) and Carya illinoinensis in China (Zhang et al. 2023). However, this is the first report of anthracnose caused by C. plurivorum on passion fruit in China. The results can provide a robust basis for scientific prevention and control of anthracnose.

First report of leaf spot disease caused by Neopestalotiopsis rosae on Lonicera caerulea L. in Heilongjiang Province, China.

Recently, interest in cultivating blue honeysuckle (Lonicera caerulea L.) for horticulture and medicinal uses has grown (Sharma and Lee 2021). Between September 2022 and September 2023, a leaf spot disease (Fig. S1) was observed on approximately 20% of 'Lanjingling' blue honeysuckles grown in a 0.18 ha field in Qiqihar city (123.43°E, 47.92°N), Heilongjiang Province, China. Infected plants displayed black leaf spots that expanded to cover the entire leaf. Small, 3 to 4 mm segments of infected tissue were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Ma et al. 2023). To induce sporulation, nine purified cultures (Fig. S2) with similar culture characteristics were finally obtained from ten infected plants and they displayed a conidia morphology consistent with Neopestalotiopsis spp., no other fungi were isolated, and the isolation frequency was 90%. Conidiomata (Fig. S3) were brown to black and distributed in concentric rings with an average size of 261.98 (60.30-451.80) µm (n = 50). The conidia (Fig. S3) were fusoid and had four septa, straight to slightly curved, with an average size of 23.48 (13.50-30.30) × 5.42 (4.50-9.30) µm(n = 50), while basal and apical cells were hyaline and the three middle cells were brown with darker septa. PCR amplification was performed with ITS1/ITS4 (White et al. 1990), EFl-728F/EF1-986R (Carbone and Kohn 1999), and Btub2Fd/Btub4Rd (Glass and Donaldson 1995) primers from the genomic DNA of the LD-330. Sequences of ITS (PP033584), TEF (PP048757), and TUB (PP048758) revealed 99 to 100% (499/500, 255/255, and 481/486) shared identity with Neopestalotiopsis rosae sequences (NR145243, KM199524, and KM199430) (Rebollar-Alviter et al. 2020). Therefore, based on morphological characteristics and molecular phylogeny, LD-330 was identified as N. rosae. Six two-year-old healthy plants of the 'Lanjingling' cultivar were selected for a pathogenicity test (Yan et al. 2023). The leaves were surface disinfected with 75% alcohol and then wiped with sterilized water three times. Three plants were inoculated with 10 ml of LD-330 conidial suspension (1 × 106 spores/ml) or with sterile water as an experimental control, respectively. All plants were in closed plastic bag, incubated in a greenhouse at 28 ℃ and 75% relative humidity (RH) under a 12-h light/dark cycle, and each experiment was performed three times (Rebollar-Alviter et al. 2020). Typical leaf spot symptoms were observed on inoculated leaves after 14 days (Fig. S4), whereas no symptoms were detected on water-treated leaves. The same pathogen was reisolated from infected leaves, displayed the same morphological and molecular traits, and was again identified as N. rosae, confirming Koch's postulate. Neopestalotiopsis rosae was previously reported on pecan (Gao et al. 2022), causing black leaf spot disease in China. To our knowledge, this is the first report of a blue honeysuckle leaf spot caused by N. rosae in China and specifically in the Heilongjiang province which has the largest blue honeysuckle cultivation area in the country. Future research should be directed toward developing comprehensive management measures.

First report of leaf spot disease caused by Cladosporium pseudocladosporioides on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) has contributed to maintaining the forest's ecological balance and remarkable frost-resistant abilities, helping it withstand extremely cold conditions (-46 °C) and a wide pH range (5 to 8) (Sharma and Lee 2021). Between September 2022 and September 2023, leaf spots were observed on approximately 30% of blue honeysuckle plants of the 'Lanjingling' cultivar grown in a 1.13 ha field in Da Hinggan Ling Prefecture (50.32° N, 124.13° E) in Heilongjiang Province, China. The leaves of the affected plants displayed black-colored spots. To identify the causal agents, 10 healthy and symptomatic leaves were randomly collected from ten healthy and infected individual plants, respectively. Small (3 to 4 mm) segments of the symptomatic tissues were immersed in 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in a paper towel, and plated on 9-cm Petri dishes containing potato dextrose agar (PDA). Ten fungal colonies developed on the PDA plates with an isolation frequency of 100% from 10 symptomatic leaves, and all colonies displayed a morphology consistent with Cladosporium spp. (Bensch et al. 2018). Cladosporium-like fungi were not isolated from healthy leaves. Dark olive-colored mycelia were observed, with straight unbranched conidiophores bearing terminal light brown-colored limoniform conidia (1.80 to 4.50 × 2.10 to 12.60 µm) and surrounded by a thin line of white mycelium (Delisle-Houde et al. 2024). To confirm this identification, PCR amplification of two representative strains LD-299 and LD-300 genomic DNA was performed with ITS1/ITS4 (White et al. 1990) and ACT512F/ACT783R (Carbone and Kohn 1999) primers. Basic local alignment search tool (BLAST) analyses of the National Center for Biotechnology Information database showed that sequences of the ITS (PP600316, PP600317) and ACT (PP624334, PP624335) all revealed 100% (493/493 nt, 493/493 nt; 181/181 nt, 181/181 nt) shared identity with Cladosporium pseudocladosporioides strain ex-type MF473195 and HM148674 (Bensch et al. 2010), respectively. Using a neighbor-joining phylogenetic analysis based on the ITS and ACT sequences, isolates LD-299 and LD-300 clustered in the same clade of C. pseudocladosporioides. Therefore, based on its morphological characteristics and molecular phylogeny, the two isolates were identified as C. pseudocladosporioides (Cosseboom and Hu 2023). A pathogenicity test was performed using nine healthy two-year-old blue honeysuckle Lanjingling plants. Three plants were inoculated with either the LD-299 or the LD-300 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control (Aydogdu et al. 2023). All plants were cultured in a greenhouse (28℃, 75% relative humidity, 12 h light and dark cycle), and each experiment was replicated three times. Typical leaf spot symptoms were first observed on the inoculated leaves after 10 days. Morphological and molecular characterization of re-isolated pathogens from the artificially infected leaves indicated that the two isolates were identical, thereby confirming Koch's postulates. Cladosporium pseudocladosporioides previously caused leaf spot disease on artichoke (Cynara scolymus) in Türkiye (Aydogdu et al. 2023). To the best of our knowledge, this is the first report of C. pseudocladosporioides causing leaf spots on blue honeysuckle in China. Blue honeysuckle production losses due to the leaf spots are critical for growers. Therefore, further focus should be given to investigate the host range and geographic distribution of C. pseudocladosporioides.

First report of Fusarium oxysporum f. sp. fragariae race 1 causing Fusarium wilt of strawberry (Fragaria × ananassa) in the Alentejo region of Portugal.

In the summer of 2023, within the Alentejo region (Portugal), a new occurrence of a plant disease of strawberry (Fragaria × ananassa) cv. 'Monterey' was observed in a Spring commercial planting. Symptoms consisted of foliar wilting, drying of older leaves, deformed and highly chlorotic leaflets, crown discoloration, plant stunting, and in some cases death. Several outbreak foci, covering nearly half a hectare, were observed within the affected farm, with almost 80% of the plants showing symptoms. Four samples,of 6 plants, were collected from 4 locations within the field. Petiole sections (1 cm) were rinsed with 0.1% Tween 20, submersed in 70% EtOH for 20 s followed by 60 s in 1% NaOCL, and then placed on Komada's medium (Komada, 1975). After incubation at room temperature in the dark for a week, white-colored fluffy mycelia grew profusely from the petioles of all samples. Colony morphology and non-septate, ellipsoidal microconidia (5.7-12.4×2.5-4.3 µm) borne on monophialides, exhibited a resemblance to Fusarium oxysporum (Leslie and Summerell, 2006). Ten single strains were obtained from different plants by single hyphal tip isolation. For molecular confirmation, a portion of the translation elongation factor 1-alpha (EF1α) was amplified by PCR using EF1/EF2 primers (O'Donnell et al., 1998). Additionally, the RNA polymerase subunit RPB2 was amplified as two contiguous fragments via primers and protocols described by O'Donnell et al., 2021. Amplicons were sequenced (GenBank Accession Nos. PP426617 - 426626, PQ058494 - 058513). Using Fusarium-ID and Fusarioid-ID databases, EF1α and RPB2 sequences were found to be more than 99% identical to published F. oxysporum type isolates and Fusarium sp. isolates in the F. oxysporum species complex (Crous et al., 2021; Wang et al., 2022). A specific F. oxysporum f. sp. fragariae (Fof) qPCR assay (Burkhardt et al., 2019) was used to determine if these isolates could be Fof race 1. A Fof race 1 isolate (MAFF305558), negative controls, and water controls were included. All ten isolates and the Fof race 1 control were positive (Ct < 30), while other Fusarium spp. used as negative controls and the water controls did not amplify. Two isolates (F.200 and F.202) and MAFF305558 (positive control) were included in a pathogenicity test on two strawberry cultivars, 'Monterey' (susceptible to race 1) and 'Fronteras' (resistant to race 1) (Dilla-Ermita et al., 2023). Each isolate was included in two independent trials. In each trial, 5 plants per cultivar were inoculated by dipping roots for 10 min in 5 × 106 conidia/mL of 0.1% water agar (WA) or in sterile 0.1% WA for the negative control plants. Each plant was then planted in a pot filled with peat. Pots were placed randomly in random positions in a growth chamber at 28/20°C and 12h photoperiod. After 8 - 10 weeks, the control plants and 'Fronteras' plants remained healthy, while the inoculated plants cv. 'Monterey' were severely wilted and/or dead. Fusarium oxysporum was re-isolated from all symptomatic plants. Recovered isolates were confirmed to be the same as the inoculated ones using the Fof-qPCR, including the same controls as above. This is the first report of Fof race 1 in the Iberian Peninsula. Given that the land was not previously used for strawberry, it is highly probable that the pathogen was introduced with the planting material originated from a Spanish nursery. In conclusion, it is imperative to implement more severe control measures in nurseries to avoid the spread of this race.

Mechanical leaf removal for improved Botrytis bunch rot control in Vitis vinifera 'Pinot gris' and 'Pinot noir' grapevines in the northeastern U.S.

Late-season bunch rot can cause substantial yield loss in grapevines grown in humid regions. Fruit zone leaf removal has been widely used to reduce bunch rot and pesticide applications through improvements in canopy microclimate and grape cluster morphology. In this study, we evaluated if mechanical leaf removal can be a valid alternative to a labor-intensive manual application by comparing pre-bloom manual (PB-MA) and mechanical (PB-ME) leaf removal. We also evaluated the effects of the timing of mechanical application, pre-bloom (PB-ME) versus fruit set (FS-ME), on fruit traits and bunch rot, caused by Botrytis cinerea. Our trials were conducted on two Vitis vinifera 'Pinot noir' and 'Pinot gris' vineyards in the northeastern US over two seasons (2017-2018). Major findings were overall consistent between cultivars and years. Leaf removal provided reductions in fruit-zone canopy density regardless of method or timing. In general, PB-ME provided similar shifts in cluster morphological traits to PB-MA, including lower number of berries per cluster, cluster compactness, and cluster weight compared to control (no leaf removal) vines. At harvest, both pre-bloom leaf removal methods equally reduced Botrytis bunch rot severity, while Botrytis bunch rot incidence in Pinot noir was lowest for PB-ME in one year and PB-MA in the next year. When comparing timing of mechanical leaf removal, FS-ME provided Botrytis bunch rot reductions similar to PB-ME, without effects on cluster weight. Thus, under our growing conditions, FS-ME was considered the best mechanical leaf removal option to help manage Botrytis bunch rot without causing undesirable yield reductions.

Sensitivity of Mucor piriformis to Natamycin and Efficacy of Natamycin Alone and with Salt and Heat Treatments Against Mucor Rot of Stored Mandarin Fruit.

Mucor rot caused by Mucor piriformis is an emerging postharvest disease of mandarin fruit in California. Natamycin is a newly registered biofungicide for postharvest use on citrus and some other fruits. In the study, baseline sensitivity to natamycin in 50 isolates of M. piriformis was determined in vitro. The mean EC50 (effective concentration to inhibit sporangiospore germination by 50%) and MIC (minimum inhibitory concentration to inhibit mycelial growth by 100%) values were 0.59 µg/ml and less than 1.0 µg/ml, respectively. Natamycin at the label rate of 920 µg/ml alone or in combination with 3% potassium sorbate (PS) or 3% sodium carbonate (SC) applied at 20 or 50°C was evaluated for control of Mucor rot on inoculated 'Tango' mandarin fruit. Natamycin alone reduced Mucor rot incidence on stored mandarin fruit from 100% among nontreated control fruit to approximately 30%, a reduction of more than 70% compared to the nontreated control, while 3% PS and 3% SC had no to little control. When applied at 50°C, natamycin and 3% PS reduced Mucor rot incidence by 65.0 and 31.2%, respectively; while natamycin in combination with 3% PS reduced disease incidence by 92.5% compared to the nontreated control after 2 weeks of storage at 5°C. This combined treatment remained effective even when the application of the treatment was delayed for 6 and 12 h after inoculation. However, the effectiveness of the treatments declined when storage was extended to 3 or 4 weeks. Natamycin can be an effective tool to control Mucor rot on mandarin fruit, and minimizing the period of extended storage could help maintain the control efficacy of natamycin.

Spatio-Temporal Spread of Grapevine Leafroll Disease in Washington State Vineyards.

The spread of grapevine leafroll disease (GLD) to vineyards planted with certified planting stock is of significant concern to grape growers. In this study, the spatial and temporal spread of GLD was examined in three vineyard blocks planted with virus-tested wine grape (Vitis vinifera) cultivars adjacent to vineyard blocks heavily infected with GLD in two geographic locations in eastern Washington State. During each season, the position of vines showing GLD symptoms was recorded in a matrix representing the planting lattice. Symptomatic vines were positive only for Grapevine leafroll-associated virus 3 (GLRaV-3), the most common virus species consistently associated with GLD in Washington vineyards. The results from multiple seasons showed a gradual increase in disease incidence over time in all three blocks. Spatial and temporal mapping of GLD indicated a disease gradient in which the highest percentage of symptomatic vines was in rows proximal to infected old blocks. Spatial autocorrelation analysis using Moran's I values suggested random patterns of symptomatic vines in the three blocks during initial years, indicating primary spread of the virus not related to infected vines within the block. Clustering at the scale of neighboring vines during subsequent years suggested secondary spread within the block. Results of quadrat-based spatial analyses of GLD incidence were compared with previously reported data obtained from California and elsewhere for an improved understanding of the dynamics of GLD spread to facilitate area-wide disease management strategies.