Sterile Toothpicks Research Articles

First Report of Stem Blight of Mungbean (Vigna radiata) Caused by Diaporthe longicolla in the United States.

Mungbean (Vigna radiata) is primarily grown in Asia and directly consumed by humans. U.S. consumers embraced mungbean as a plant-based protein in vegan eggs and meat substitutes. New cultivars are being developed for American farmers because of the crop's tolerance to heat and drought, and its adaptability to current farming infrastructure. Mungbean's short season complements various cropping systems such as intercropping, alternative cropping, and green manure. With rotations and inclusion with soybean systems, there is a concern about the overlap of common pathogens for soybean and mungbean. During August 2022 when mungbeans reached full maturity (growth stage R6), reddish-brown and necrotic stem lesions with linear rows of black pycnidia were observed on Berken and OK2000 cultivars at fields located in Hancock County, IA and Story County, IA in the United States. Pycnidia measured 0.5-0.6mm in length. Disease incidence was approximately 10% of plants in Hancock County, IA and less than 3% of plants in Story County, IA. Pycnidia from 16 plants were excised and immersed in a 0.5% NaOCl solution for 1 min, rinsed with autoclaved distilled water, and placed onto potato dextrose agar (PDA). Eighteen isolates were hyphal tipped and grown on PDA and were stored at 25°C. Isolates were then visually identified by culture and conidia morphology (Hobbs et al. 1985, Santos et al. 2011). Colonies were cream to white, dense, and floccose. Large black stromata were formed in a concentric pattern or scattered; alpha conidia were ellipsoidal. Template DNA for PCR amplification of the internal transcribed spacer region of the nuclear ribosomal DNA operon (ITS) and the beta-tubulin gene (TUB) was extracted from 18 isolates by scraping mycelia with a sterile pipette tip and transferring it into 50 ul of PrepMan Ultra Sample Reagent (Applied Biosystems, Foster City, California, USA). Fungal primers were ITS1 and ITS4 (White et al. 1990) and Bt-2F/Bt-2R (Udayanga et al. 2014). Sequences of isolates obtained from fields in both counties were identical, providing no species diversity. GenBank accession numbers for the ITS region were PP105598 and PP105599; PP108254 and PP108255 for TUB sequences. BLAST results showed the ITS 550/550 base pairs with type specimen D. longicolla ATCC 60326 GB NR_144924 and the TUB 446/446 base pairs with type specimen D. longicolla ATCC 60325 GB KJ610883. Thus, the isolates were identified as Diaporthe longicolla (Hobbs) J.M. Santos, Vrandecic & A.J.L. Phillips based on morphology and molecular characters (Santo et al 2011; Udayanga et al. 2014). To confirm the pathogenicity of the D. longicolla isolates, twenty mungbean plants (cv. Berken and OK2000) were grown in the greenhouse at 85% RH and 16hr light for 20 days. Inoculum was prepared by placing sterile toothpicks on 1/3 PDA with a single representative isolate from each field location for 21 days (Ghimire et al. 2019). Mungbean plants were grown in a 10cm-by-10cm pot containing a greenhouse professional growing mix (Sungrow, Agawam, Massachusetts, USA) and grown for 30 days post emergence. After 12 days of growing, a 3mm segment of the infested toothpick was inserted into a stem wound below the first trifoliate and sealed with parafilm. A sterile toothpick was inserted into the control plants. After 14 days, red lesions extended downward 1 to 3 cm from the inoculation site, and white mycelial was present in the wound. At 21 days red lesions spanned 3 to 9 cm upward and downward from the inoculation site. Pycnidia were present on collapsed stem tissue, and leaves became chlorotic. Damage was limited to 2mm from the mock-inoculation site, with no discoloration in the control plants. Symptomatic tissues were plated and compared to the original isolates. Alpha conidia were ellipsoidal with the base end rounded. To our knowledge, this is the first report of Diaporthe longicolla causing disease on mungbean within the U.S. and worldwide. The presence of this disease in two locations suggests the potential for Diaporthe longicolla to be a serious disease of mungbean in the future.

Heliconia subulata, a New Host of Colletotrichum tropicale Causing Anthracnose in China.

Heliconia subulata is a common ornamental plant, it has been widely planted in southern China for greening parks, roads, and residential areas. H. subulata plants with spots on their leaves were observed in East Coast Wetland Park (18°16'53.37″N, 109°30'19.36″E), Sanya City, Hainan Province, China on Aug. 31, 2023. The symptoms of the leaves are irregular gray-white, spots, that develop into brown and black, with yellow halos at the disease-health junction. Following an on-the-spot investigation, it was found that the incidence of the disease was 40 to 50%. The leaves were disinfected with 70% ethanol for 1 min, rinsed with sterile water 3 times, disinfected for 1 min with 0.1% HgCl2, rinsed with sterile water 3 times, dried, put on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. The red conidia pile was selected from the culture, dispersed in sterile water and diluted to 20 μL containing 1 to 2 conidia. After absorbing 20 μL spore suspension for many times and inoculating it on the new PDA plate, five pure cultures of single spore, J-1-1 to J-1-5, were obtained. After 7 days of growth, the colonies were grayish aerial mycelium on the front and light orange conidia on the reverse. The white aerial mycelia, conidia, acervulus, and appressorium were observed (Supplementary Fig. S1). The morphological characteristics showed that the isolate had the same characteristics as the previously described Colletotrichum spp. (Wang et al. 2021). The genomic DNA of isolates J-1-1 and J-1-5 were extracted by Fungal DNA Kit (OMEGA bio-tek, Guangzhou, China). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and β-tubulin 2 genes (TUB2) were amplified by primers ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b, respectively (Weir et al. 2012). Based on sequencing and gene sequence alignment analysis, it was found that the consistency between the ITS sequences of isolates J-1-1 and J-1-5 was 99.82%. The consistency between GADPH and TUB2 sequences was 100%. The gene sequences of isolates J-1-1 and J-1-5 were submitted to GenBank with accession numbers PP455510/PP455511 (ITS), PP510210/PP510211 (GADPH) and PP510212/PP510213 (TUB2) respectively. Based on the BLAST analysis, the three sequences were more than 99% identical to those of theC.tropicalestrain FC1 (ITS: MT192648, GAPDH: MT155819, TUB2: MT199874; Duan et al. 2022). A phylogenetic tree was constructed by MEGA 11 based on the ITS, GADPH, and TUB2 gene sequence by the maximum-likelihood method. The results showed that the isolates J-1-1 and J-1-5 were clustered with C. tropicale CBS:124949 (Supplementary Fig. S2). Based on morphological and molecular biological analysis, two isolates were identified as C. tropicale. To further test the pathogenicity of isolates J-1-1 and J-1-5, spore suspensions (1×106conidia/mL) were prepared and 20 μL spore suspensions were inoculated on the leaves of healthy H. subulata potted plants stabbed with sterile toothpicks. Three leaves were inoculated in each treatment, and sterile water was inoculated as a control. The treated plants were placed in an incubator with a temperature of 28℃, relative humidity of 90%, and light/dark (12h/12h). After 15 days, the spore suspension treatment showed the same symptoms as the naturally diseased H. subulata plants in the field, but the leaves treated with sterile water were not infected (Supplementary Fig. S1). The morphology of the isolates obtained from diseased leaves was the same as that of isolates J-1-1 and J-1-5 on the PDA plate. To our knowledge, this is the first report of H. subulata, a new host of C. tropicale causing anthracnose in China.

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

First Report of Anthracnose on Wurfbainia villosa var. villosa Caused by Colletotrichum gloeosporioides in China.

Wurfbainia villosa var. villosa is a traditional Chinese herbal medicine under the family Zingiberaceae, and its ripe fruits (called Fructus Amomi) are widely used clinically for the treatment of gastrointestinal disorders (Yang et al. 2023; Chen et al. 2023). In September 2023, plants of W. villosa var. villosa exhibited anthracnose-like symptoms on leaf with a disease incidence of 35% (n = 100 investigated plants) in an approximately 90 m2 field in Guangning, China (N23°42'51.70″, E112°26'35.75″). Light yellowish-green spots (~2 mm diameter) initially appeared on the infected leaves, gradually formed sub-circular or irregular spots, then fused and expanded, resulting in wilting of the leaves. To identify the causal agent, 10 symptomatic leaves were collected and transferred to the laboratory. The symptomatic leaf samples were surface sterilized in 0.5% NaClO for 2 min, and in 70% ethanol for 30 s, then washed three times with sterile water and air-dried on sterile filter paper. The leaf tissues were placed on potato dextrose agar (PDA) medium containing 100 μg mL-1 of ampicillin (Sigma-Aldrich, St. Louis, MO) and incubated for 7 days at 28°C in darkness. Nine isolates with similar colony morphology were isolated from the 10 plated leaves. Three representative isolates (GNAF03, GNAF06, GNAF09 with approximately 3.5 cm in diameter after 3 days of incubation) appeared gray to dark brown with dense aerial hyphae at the front and gray to black colonies on the reverse of the plates. Conidia were cylindrical and measured 21.2 to 29.3 μm long × 7.1 to 9.6 μm wide (n = 50). Appressoria were formed by the tips of germ tubes or hyphae and were brown, ellipsoid, thick-walled, and smooth-margined, measuring 10.2 to 12.3 μm long × 6.4 to 8.2 μm wide (n = 50). Morphologically, the fungal isolates resembled Colletotrichum sp. (Weir et al. 2012). For molecular analysis, genomic DNA was extracted from fresh mycelia of the three isolates, and the primers ACT-512F/ACT-783R, CL1/CL2A, GDF/GDR, and ITS1/ITS4 were used to amplify partial regions of rDNA-ITS, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions, respectively (Weir et al. 2012). The resulting sequences with more than 99% nucleotide identity to C. gloeosporioides were submitted to GenBank (accession numbers PP552725, PP552726, and OR827444 for ACT; PP552727, PP552728, and OR827443 for CAL; PP552729, PP552730, and OR827445 for GAPDH; PP549996, PP549999, and OR841394 for ITS). A phylogenetic tree was generated by the maximum likelihood method using the concatenated sequences of ACT, CAL, GADPH, and ITS by Polysuite software (Damm et al. 2020). Based on morphological and molecular analysis, the three isolates were characterized as C. gloeosporioides. The pathogenicity of the GNAF09 isolate was assessed on W. villosa var. villosa seedling leaves inoculated by spraying with 40 μL of conidial suspension at 106 conidia mL-1 or wounded with a sterile toothpick then inoculated with mycelial agar plugs (5 mm diameter). Control leaves were inoculated with 40 μL of sterile distilled water or agar plugs without mycelia. The inoculated plants were placed in a humid chamber at 28°C with 80% humidity and a 12 h light-dark photoperiod. Symptoms similar to those seen on naturally infected leaves were observed on all inoculated leaves after 7 days inoculation. Re-isolation was performed from 80% of the inoculated leaves and isolates were confirmed as C. gloeosporioides morphologically, confirming Koch's postulates, and by sequencing the ACT, CAL, GADPH, and ITS regions. The control groups remained asymptomatic. In previous studies, C. gloeosporioides has also caused anthracnose on Chinese medicinal plants, including Baishao (Radix paeoniae alba) (Zhang et al. 2017) and Rubia cordifolia L. (Tang et al. 2020). To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. villosa var. villosa in China. The results of our report serve as valuable references for further research on this disease.

Bisifusarium lunatum causing cladode soft rot in pear cactus (Nopalea cochenillifera L. Salm-Dyck) in Brazil.

Cactus pear var. miúda (Nopalea cochenillifera L. Salm-Dyck) is an important crop for the Northeast region of Brazil, composing one of the main sources of animal feed. By April 2021, cladode rot caused death of several cactus pear plants in a production area located in Itaporanga, Paraíba state, Brazil (7°21'55.35" S and 38°11'38.68" W). The infected cladodes showed brown circular necrotic spots, and soft rot with perforations that extended throughout the cladode, followed by tipping over and death of the infected plants. The incidence of the disease ranged from 10 to 30% of the plants. Bisifusarium strains were isolated and cultured on potato dextrose agar (PDA) and syntetic-nutrient-poor-agar (SNA). The colonies showed purple color on PDA. On SNA, macroconidia (n = 100) were abundant, hyaline, slightly falcate, three-septate, measuring 11.0-23.1 x 2.3-4.1 μm. Microconidia (n = 100) were oval, generally aseptate, measuring 4.1-8.7 x 2.3-3.0 μm. Conidiogenic cells formed into short monophialides. Chlamydospores were not observed. According to these morphological features, the pathogen was initially identified as Bisifusarium lunatum (Gryzenhoutm et al. 2017). For further confirmation of the identification, the partial sequences of translation elongation factor 1-alpha (TEF1-α) and the second largest subunit of RNA polymerase II (RPB2) genes were sequenced for a representative isolate (CMA 34: GenBank accession no: TEF1-α: OR536502; and RPB2: OR553509) and compared to other Bisifusarium species from GenBank database. Subsequently, it was subjected to a phylogenetic analysis of maximum likelihood including previously published sequences. According to BLAST searches, the TEF1-α and RPB2 sequences were 99% (637/640 nt) and 100% (312/312 nt) similar to B. lunatum (COUFAL0213: TEF1-α (MK640219), and RPB2 (MK301291)), respectively. The isolate was also clustered in a clade containing the ex-type of B. lunatum with 100% support (SH-aLRT and UFboot), being confidently assigned to this species. The pathogenicity test was performed after Medeiros et al. (2015), by using healthy two months old cactus pear seedlings (n = 10) cultivated in a greenhouse. Sterile toothpicks were distributed over colonies of the representative isolate grown on PDA at 25 ± 2 °C for seven days. Seedling cladodes were stuck with the toothpicks, moistened with sterile water and covered with transparent plastic bags for 24h, thus simulating a humid chamber. Following three months, all control plants (stuck with sterile toothpicks) remained healthy, while those inoculated with the representative isolate exhibited rot symptoms. This test was performed twice. B. lunatum was reisolated from symptomatic cladodes and identified as previously described, thus fulfilling the Koch's postulates. To our best knowledge, this is the first report of B. lunatum causing soft rot on N. cochenillifera in Brazil. Besides N. cochenillifera, this species was also reported on Opuntia ficus-indica in India (Gryzenhoutm et al., 2017), which raises concern regarding its ability to infect other forage sources for cattle feed in Brazilian semiarid regions. The present study highlights that the precise identification of B. lunatum is a key factor to adjust control strategies and management of the disease to prevent the spread of this disease to prevent its spread to other crops.

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 Anthracnose on Banana Peppers Caused By Colletotrichum scovillei in New York.

In early August 2023, a disease outbreak on hot banana peppers (Capsicum annuum cv. Golden Dagger) was reported in Cattaraugus County, New York (NY). Disease incidence was at least 60%. Affected developing and mature fruit had at least one tan, soft, sunken lesion with salmon-colored spore masses surrounded by brown, necrotic margins. Microscopic observation of the lesions identified acervuli and setae typical of Colletotrichum spp. Isolations were made from these lesions by spreading conidia from the acervuli on 2% water agar (WA) + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred onto clarified V8 juice agar (CV8) and incubated at 20°C. The isolation frequency was 100% and a total of six isolates were obtained: Coll23Pep001, Coll23Pep003, Coll23Pep005, Coll23Pep007, Coll23Pep008, and Coll23Pep010. After 10 days, colonies were subcultured to potato dextrose agar (PDA) and CV8. On PDA, colonies appeared off-white to dark gray with sparse aerial mycelia. On CV8, the colony was pale gray with acervuli and orange-colored spore masses in the center. Conidia were hyaline, smooth and fusiform to round, and tapered at both ends. Mean conidial dimensions (n = 20) were 20.2 (13.75 to 25) µm long × 4.7 (3 to 6.25) µm wide. To confirm the identity of the isolates, DNA was extracted, and PCR performed to amplify the internal transcriber spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), and actin (ACT) (primers ACT-512F/ACT-783R; Carbone and Kohn 1999) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (primers GDF1/GDR1; Guerber et al. 2003). Pairwise alignment of the sequences showed all isolates had 100% similarity to C. scovillei ex. holotype CBS 126529 (Damm et al. 2012). Sequences from all isolates were deposited in GenBank with accessions PP556967 to PP556972 (ITS), PP565766 to PP565771 (ACT), and PP565772 to PP565777 (GAPDH). For pathogenicity testing, all isolates were grown on CV8 at 20°C in the dark for 10 days. Conidia were harvested by flooding the plates of each isolate with sterile distilled water and filtering the suspension through a double layer of cheesecloth. The concentration of the conidial suspension was adjusted to 5 × 105 spores per ml. Pathogenicity of the six isolates was tested on banana pepper fruit by using a sterile toothpick to pierce the skin at the two opposite ends. A droplet (10 µl) of the conidial suspension was placed on each wound. The same number of fruit were inoculated without wounding, and non-inoculated control fruit received a droplet of sterile distilled water (either wounded or unwounded). The experiment was repeated twice. All fruit were placed in a humid box at room temperature for 7 days. All wounded and inoculated fruit developed sunken lesions filled with salmon-colored conidial masses. Disease did not occur on the unwounded, inoculated fruit nor the non-inoculated controls. C. scovillei was recovered from all inoculated fruit by reisolating onto CV8 media and isolates had similar morphology and conidial dimensions to the original isolates. To the best of our knowledge, this is the first report of C. scovillei causing anthracnose on pepper in NY. C. scovillei has been reported in South Carolina (Toporek and Keinath 2021), Brazil (Caires et al. 2014), eastern Asia (de Silva et al. 2019), and Kosovo (Xhemali et al. 2023). The pathogen is particularly aggressive on pepper and poses substantial threats to pepper production around the world.

First Report of Rhizoctonia solani AG-4 HG-I Causing Root and Basal Stem Rot on Roselle (Hibiscus sabdariffa) in Mexico.

Roselle (Hibiscus sabdariffa L.) is a crop of economic importance, refreshing drinks are prepared from its calyces, it is also attributed to antioxidant, antibacterial, and antihypertensive properties (Da-Costa-Rocha et al. 2014). In November 2022, in municipality of Iguala (18.355592N, 99.548546W, 749 m above sea level), Guerrero, México, roselle plants of approximately 1.5 months of age with basal rot were detected under greenhouse conditions. The symptoms consisted of wilting, yellowing, and root and stem rot with constriction in the base of the stem. The symptoms were detected in approximately 15% of plants at the operation. From symptomatic tissue, cuts were made into approximately 0.5 cm pieces, sterilized with 2% NaClO, washed with sterile distilled water, transferred to PDA medium amended with 50 mg/liter of Chloramphenicol, and incubated in the dark for four days at 28 °C. Rhizoctonia-like colonies were consistently obtained, and nine isolates were selected and purified by the hyphal-tip method. After four days, isolates developed a mycelium was light-white that became brown with age. Right-angled hyphal branching was also observed, in addition to a slight constriction at the base of the branches. In some older cultures, numerous dark brown sclerotia were observed. They were multinucleate cell with three to eight nuclei and measured from 1 to 2 mm in diameter. Together these characteristics were consistent with the description of Rhizoctonia solani Kühn (Parmeter 1970). The anastomosis group (AG) was confirmed by amplifying the ITS region with the primers ITS1 and ITS4 (White et al. 1990) of the RIJAM3 and RIJAM5 strains. The sequences were deposited in GenBank (Nos. OR364496 and OR364497 for RIJAM3 and RIJAM5, respectively). BLAST analysis, both isolates indicated 99.7 identity to R. solani AG-4 HG-I (GenBank: KM013470) strain ICMP 20043 (Ireland et al. 2015). The phylogenetic analysis of AGs sequences allowed assignment of isolates RIJAM3 and RIJAM5 to the AG-4 HG-1 clade. A pathogenicity test was performed on 20 one-month-old roselle plants. Mycelium of RIJAM3 isolate was inserted into the base of the stem with a sterile toothpick. As a control, a sterile toothpick with no mycelium was inserted in ten healthy plants. Additionally, 50 eight-day-old seedlings were inoculated by placing a 5-mm diameter agar plug colonized with mycelium of RIJAM3 at the base of the stem 10 mm below the soil surface. As control treatments, uncolonized PDA plugs were deposited at the base of 25 seedlings. The inoculated plants were incubated in a greenhouse with an average temperature and relative humidity of 28°C and 85%, respectively. Following inoculation, symptoms similar to those observed in the original outbreak were observed in plants after six days and only after four days in seedlings. In both experiments, the control plants and seedlings remained asymptomatic. R. solani was re-isolated from plants and seedlings, complying with Koch's postulates. The pathogenicity testing was repeated twice, with concordant results. In Nigeria and Malaysia R. solani was reported to seedling death to cause seedling dieback in roselle (Adeniji 1970; Eslaminejad and Zakaria 2011). In México R. solani AG-4 has been previously reported in crops of potato, chili and tomato (Montero-Tavera et al. 2013; Ortega-Acosta et al. 2022; Virgen-Calleros et al. 2000). To the best of our knowledge, this is the first report of R. solani AG-4 HG-I as a causing of root and basal stem rot on roselle in Mexico. This research provides information essential for informing the management of this disease, and may help design measures to prevent the spread of the pathogen to other regions.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Peanut in Chongqing, China.

In July 2022, dieback and discoloration were detected on infected stems of peanut in Qijiang District of Chongqing (106.56°E,29.41°N), China, with an incidence up to 5%. These peanut stems had disease symptoms typical of anthracnose with irregular gray-brown spots with dark brown edges, sunken, and necrotic. High temperature and high humidity were favorable for the growth of the pathogen. To isolate the pathogen, we collected 10 typical infected peanuts and cut one piece from each of symptomatic stems, surface sterilized with 0.5% NaClO for 1 min, and 75% ethanol for 30 s, then rinsed three times with sterile distilled water and dried on sterilized filter paper. These pieces were incubated on potato dextrose agar (PDA) at 25°C in the dark. Pure cultures were obtained from hyphal tips of each colony. It was found that isolates with the same colony morphology were isolated from each infected stem. A representative isolate (L7) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. The colonies appeared white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, 13.4 to 18.8 × 4.2 to 5.8 μm (n=50). Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Cannon et al., 2012). For molecular identification, genomic DNA was extracted using a CTAB method and partial sequences of β-tubulin (TUB2), actin (ACT) genes, chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced using primers T1/T2, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and GDF1/GDR1, respectively (Damm et al., 2012; Dowling et al., 2020). Using the BLAST, TUB2, ACT, CHS and GAPDH gene sequences (GenBank accession No. OR714793, OP168707, OP168708 and OR714794, respectively) were100% (429 bp out of 429 bp), 99.22% (256 bp out of 258 bp), 99.64% (276 bp out of 277 bp) and 100% (253 bp out of 253 bp) identical to C. gloeosporioides CBS:112999 (JQ005587, JQ005500, JQ005326, and JQ005239), respectively. Using Neighbor-Joining algorithm, phylogenetic analysis was conducted based on the concatenated sequences of published TUB2, ACT, CHS and GAPDH genes. The identified isolate (L7) was closely related to C. gloeosporioides. To evaluate pathogenicity, the stems of ten peanut (Zhonghua12) seedlings (2 weeks) were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) or 20 μl of conidial suspension (105/ml) were inoculated. Non-colonized agar plugs or 20 μl of sterile distilled water were treated as control. After inoculation, the peanuts were kept in a moist chamber at 28°C with 80% humidity in the dark for 24 h, and subsequently transferred to the moist chamber with 12 h light and darkness cycle for 6 days, similar symptoms were observed on all inoculated peanuts. Controls remained asymptomatic. C. gloeosporioides was reisolated from the diseased stems and confirmed using morphological features and sequence analysis of TUB2, ACT, CHS and GAPDH. Anthracnose caused by C. truncatum and C. fructicola has been reported on peanut leaves in China (Gong et al., 2023; Yu et al., 2019). To our knowledge, this is the first report of anthracnose on peanut stem caused by C. gloeosporioides in Chongqing. Our report will provide crucial information for studying on epidemiology and management of this disease.

First Report of Pantoea ananatis and Pantoea eucalypti causing onion leaf blight and bulb decay in Central Chile.

Nearly 5,400 hectares of long-day onions (Allium cepa) are cultivated in the Central Zone of Chile (UTM 33°3´S to 37°24´S). During summer 2021-22 and 2022-23 (December to February), followed by high temperatures (around 35°C) and high humidity conditions, symptoms showing yellowing, soft rot, blight on old leaves, necrotic tips, and soft bulbs were observed. The affected plants were observed in "spots" into the fields, and the incidence reached 5-10%. The severity was high, and 70-80% of the affected plants died. Symptomatic plants from different fields from the Coquimbo, Metropolitan, O´Higgins and Maule regions were sampled. Isolations were made using casamino-acid peptone glucose (CPG) agar medium (Schaad, 2001). Yellow-pigmented, circular to irregular shaped colonies were observed. Molecular identification was carried out by partial 16S rRNA gene sequencing, resulting in the identification of 18 isolates of Pantoea spp. from twelve different fields. Selected strains were biochemically analyzed using the GEN III BIOLOG microtest system (Hayward, CA) and were identified as Pantoea spp. BLAST analyses of the 16S rRNA sequences (602 nt) of selected strains (GenBank Accession No. OR527817 to OR527819) against the NCBI Database resulted in the identification of Pantoea species with 100% coverage and 100% identity. To determine the Pantoea species of each strain, housekeeping gene gyrB (Delétoile et al. 2009) was amplified and sequenced (GenBank Accessions No. OR544061 to OR544063). BLAST analysis (802 nt) of selected strains resulted in 100% coverage and 100% identity, identifying three different species: P. ananatis, P. eucalypti, and P. agglomerans. Pantoea species were isolated from both leaves and bulbs and no more than one species was observed per field. Pathogenicity assays in onion plants and bulbs were performed based on the methodology described by Asselin et al. (2018). Five onion plants cv. Cimarron of thirteen-week-old were inoculated by wounding an external leave with a sterile toothpick previously immersed in a bacterial suspension at ~ 108 CFU/mL and maintained at 26-28°C for 30 days in high humidity conditions. Control plants were inoculated with sterile water. Plants inoculated with Pantoea spp. showed chlorosis, soft rot, and necrosis mainly in older leaves, as observed in the field, while negative control plants remained healthy. Pantoea spp. were re-isolated from the inoculated onion plants. Toothpicks dipped in the inoculum were stuck 4 cm into the shoulders of onion bulbs and incubated at 26°C for 20 days. Water was inoculated as a negative control. At the end of the incubation period, the bulbs were opened longitudinally across their inoculation sites and shrunken, brownish, watery scales were observed. Pantoea species have been previously described as causing leaf blight of onions in Georgia and Michigan in the USA and South Africa, Brazil, and Uruguay (Hattingh and Walters 1981; Gitaitis and Gay 1997; Edens et al. 2006; Tho et al. 2015; De Armas et al. 2022; Rosende et al. 2022). P. agglomerans was recently reported in Chile (Sepúlveda et al. 2023), but this is the first report of P. ananatis and P. eucalypti affecting onions in central Chile. This detection is an alert call for the onion's growers and exporters in Chile for upcoming seasons, where conditions predisposing to disease may continue to occur. It is crucial to continue analyzing the factors that caused the appearance of this new disease in onions.

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

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

First report of Phyllosticta capitalensis causing brown spot disease on guava fruits (Psidium guajava) in Mexico.

The guava (Psidium guajava L.) is a plant native to the tropical region of America. In Mexico, the area established with guava cultivation is 20,525 ha (SIAP 2021). Guava is commonly consumed as fresh fruit, being rich in nutrients such as vitamins and minerals (Murthy et al. 2020). During October 2020, in the municipality of Cocula (18.207835N, 99.670322W, 595 m above sea level), Guerrero, Mexico, severely infected immature guava fruits were observed. The incidence of disease in 150 sampled fruits was 12%. Were collected fifteen symptomatic fruits. The symptoms were circular to irregular dark brown spots that varied in size (0.5 to 2.5 cm). From symptomatic fruits, tissues were cut approximately 3 x 3 mm and disinfested with 1% NaOCl, washed three times with sterile distilled water, and transferred to PDA medium amended with streptomycin and tetracycline, and incubated at 28°C. Developing colonies were retransferred to new culture PDA medium, and purified by hyphal tip technique. Two representative isolates (PHYGUA7 and PHYGUA3) were selected for morphological and molecular characterization. After 15 days in PDA at 28°C in an incubator, colonies were flat, irregular, granular and greenish gray, pycnidia were black, granular, and grouped. The conidia were hyaline and ellipsoid, unicellular and smooth-walled, 7-11×5-6.5 μm (n=50), these characteristics were consistent with those described for the fungus Phyllosticta capitalensis (Wikee et al. 2013). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), the actin gene (ACT), and the translation elongation factor 1-alpha (EF-1α) gene, using primers ITS1 and ITS4, ACT-512F/ ACT-783R, and EF1-728F/EF1-986R, respectively (White et al. 1990; Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (PHYGUA7: OP810947, PHYGUA3: OP810948 for ITS, PHYGUA7: OP819845, PHYGUA3: OP819846 for ACT, and PHYGUA7: OP819847, PHYGUA3: OP819848 for EF-1α). Phylogenetic analysis using maximum likelihood concatenated sequences ITS, ACT and EF-1α with MEGA X, indicated that PHYGUA7 and PHYGUA3 isolated grouped with P. capitalensis (CPC 18848 type strain). For pathogenicity test of P. capitalensis, 15 healthy immature fruits in a field experiment in the fruits on the trees, and 15 healthy mature guava fruits (detached fruits) were superficially disinfected with 70% ethanol, wounded with a sterile toothpick, and inoculated at two equidistant points by inserting PHYGUA7 isolate mycelium. As a control treatment, 10 healthy immature fruits and 10 healthy mature fruits were only injured with a sterile toothpick. After 3 days symptoms were observed in mature fruits and numerous dark pycnidia developed, and seven days later symptoms were observed in immature fruits in all the points inoculated with the PHYGUA7 isolate, similar to the symptoms observed in the field. The control fruits remained asymptomatic. The fungus P. capitalensis was re-isolated from inoculated fruits, thus confirming Koch's postulates. In Mexico P. capitalensis has been reported in Mangifera indica, Epidendrum sp., and Schomburgkia tibicinis (Farr and Rossman, 2022). In Egypt and China P. capitalensis causes black spot on guava fruits (Arafat 2016; Liao et al. 2020). To our knowledge, this is the first report of P. capitalensis as the cause of brown spot on immature guava fruit in Mexico. This research provides relevant information to the design of disease management strategies.

First report of Dickeya dadantii causing bacterial soft rot of Scindapsus pictus in Taiwan.

Scindapsus pictus (satin pothos or silver vine) is an evergreen climbing plant belonging to the Araceae family, subfamily Monstereae (Bown, 2000), which is also cultivated as a foliage ornamental (Masnira et al. 2019). In September of 2022, soft rot symptoms were observed on potted S. pictus plants grown in a greenhouse in Nantun District, Taichung, Taiwan, in which soft rot of another aroid (philodendron) has also been reported (Wu et al. 2023). The symptoms appeared on the petioles and most of them tended to extend to the leaf blades; the colors of leaf lesions ranged from dark brown to gray (Fig. S1). Some 70% of the plants in the greenhouse showed similar symptoms and losses were estimated to be 15-30%. Four symptomatic plants were sampled. Macerated tissues from rotting petioles were soaked in 10 mM MgCl2 and observed under a light microscope (Nikon, Japan) at 400 x magnification. Motile, rod-shaped bacteria were observed, and 1-2 loopfuls of undiluted sample suspension were streaked onto nutrient agar (NA; Gibco, USA). After culturing at 28°C for 1 day, all samples yielded round, creamy-white colonies (0.9 mm in diameter) and from each of the four samples a pure culture was obtained (Spi1-Spi4). All isolates exhibited oxidative and fermentative metabolism of glucose (Schaad et al. 2001). They caused pitting on crystal violet pectate agar, induced maceration on potato tuber and were tested positive for phosphatase activity and indigoidine production (Lee and Yu 2006; Schaad et al. 2001). Polymerase chain reaction tests using Dickeya-specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon (0.5 kb) in extracted DNA samples of all isolates. Identification of the strains was achieved by amplifying and sequencing fragments of the housekeeping genes gyrB, recN, dnaA, dnaJ, and dnaX (Marrero et al. 2013); the lengths of the five gene fragments analyzed were 822, 762, 720, 672, and 450 bp, respectively (accession nos. OP985528-OP985532). The five sequences were concatenated for every isolate; the resulting 3,426 bp sequences were aligned with ClustalW and found to be identical. A maximum-likelihood analysis was conducted using the 3,426-bp sequences and those of known Dickeya species' type strains. Spi1 to Spi4 clustered with D. dadantii subsp. dieffenbachiae NCPPB 2976T and D. dadantii subsp. dadantii CFBP 1269T (Fig. S2) with sequence identities of 98.4 and 98%, respectively. To fulfil Koch's Postulates, stab inoculations of the four isolates into the petioles of cutting propagated, 38-day-old S. pictus plants (3 plants per isolate) were conducted using sterile toothpicks. The amounts of bacteria used was approximately 106 cfu per toothpick; the bacterial loads were estimated by suspending the cells in 10 mM MgCl2 and spread-plating diluted suspensions on NA. Sterile toothpicks were used as control. All tested plants were sealed in plastic bags (containing wet paper towel) and kept in a growth chamber (28°C; 12-h photoperiod). After 1 day, all isolates induced soft rot symptoms resembling those observed under natural conditions in the greenhouse. Bacteria were re-isolated, and they all shared the same dnaX sequence with strains Spi1 to Spi4. This is the first report of S. pictus affected by D. dadantii in Taiwan. Further investigation is needed to determine whether Spi1-Spi4 belong to D. dadantii subsp. dieffenbachiae. Dickeya dadantii has been found infecting different aroids (Lee and Chen 2021; Lin et al. 2012). The species has also been reported in Taiwan on poinsettia (Wei et al., 2019) and philodendron (Wu et al. 2023). Because these plants are often grown closely in the same facilities, growers should be wary of D. dadantii's spread among these plants. Reduction of environmental humidity and avoiding overhead irrigation may be effective in preventing the pathogen's transmission.

First Report of Anthracnose on Chili Pepper Caused by Colletotrichum sojae in Hebei Province, China.

China is the largest chili pepper producing country, and Hebei Province stands out as the forth with planting area at about 1500 km2 in China. Pepper (Capsicum annuum L.) is susceptible to Colletotrichum spp. infection during its growth, which seriously affects production yield and quality. In September 2020, widespread anthracnose was observed on pepper in Hebei (115.48° N, 38.77° E), China. Necrotic lesions on pepper fruits were suborbcular, sunken, with acervuli arranged in the middle of lesion (e-Xtra 1A). To perform fungal isolation, small tissue with 0.3 cm2 in size at the symptomatic tissue margin was surface disinfested with 75% ethanol for 10 s, and 0.1% HgCl2 for 40 s, then washed three times with sterile ddH2O. Fragments were placed on potato dextrose agar (PDA) amended with 100 mg·L-1 chloramphenicol and incubated at 28 ºC under darkness for 4 days. One of the strains of Colletotrichum spp., named HQY157, was purified by single-spore isolation, then used for morphological characterization, phylogenetic analysis, and pathogenicity tests. Colonies presented light grey aerial mycelium, occasionally mixed with gray-black strips, and the reverse was similar to the surface on PDA (e-Xtra 1B). Conidia were smooth-walled, aseptate, straight with obtuse to slightly rounded ends, 17.3-28.5 × 3.1-7.4 μm (n=50) (e-Xtra 1C). For molecular identification, the internal transcribed spacer (ITS) region, partial sequences of actin (ACT), β-tublin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS) were sequenced using the specific primers (Weir et al. 2012). Sequences were deposited in GenBank with the following accession numbers OM317600-OM317604. A Maximum-Likelihood phylogenetic tree was constructed, based on the concatenated sequences (ACT, CHS, GAPDH, TUB, and ITS) of HQY157 and other closely matching Colletotrichum species obtained from GenBank, by using MEGA-X. It showed that HQY157 was grouped with the C. sojae with bootstrap values of 100% (e-Xtra 2). To confirm the pathogenicity, surface-sterilized healthy pepper fruits and healthy fruits with wounds (deal with a sterile toothpick after surface-sterilized) were then inoculated with 2 μL of conidial suspension (106 conidia/mL). The fruits inoculated with 2 μL sterile distilled water were taken as negative controls. After inoculation, the fruits were kept in a plastic box with sterilized filter paper moistened with sterilized water, and maintained at 25°C in the dark. The experiment was repeated three times. Anthracnose symptoms were observed 7 days after inoculation on the wounded pepper fruits, whereas the unwounded and negative control fruits remained symptomless (e-Xtra 1D). Colletotrichum sojae was re-isolated from the infected pepper fruits and identified by morphological and molecular analysis, fulfilling Koch's postulates. Colletotrichum sojae occurs mainly on Fabaceae plants such as Glycine max, Medicago sativa, Phaseolus vulgaris, and Vigna unguiculata (Damm et al. 2019, Talhinhas and Baroncelli, 2021), and Panax quinquefolium (Guan et al. 2021). To our knowledge, this is the first report of C. sojae causing anthracnose on pepper in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this chili pepper disease.

First Report of Lasiodiplodia theobromae Causing Dieback Symptoms on Plantain (Musa AAB subgroup) in Nigeria.

Bananas (banana and plantains) rank sixth among staple food crops (FAO 2018), with production challenged by biotic factors, mainly fungal diseases that may cause a total loss in some orchards (Jones 2018). In April 2017, dieback symptoms (progressive blackening and necrotic aerial plant parts, leaves, fruits and peduncles) were observed on plantain (Musa AAB subgroup), in Onne, Rivers State, Nigeria (4°42'55.4012″N, 7°10'35.92128″E). Diseased plants (n=112) were either wilted with blackened necrotic areas, or dead (Fig. S1). Nearly 10% of the plants had blackened pseudostems and fruits with slate gray to black internal tissues when sliced (Fig. S1) and black, erumpent pycnidia were observed on diseased fruits. A fungal species was consistently isolated when surface disinfected pieces of diseased samples were cultured on PDA plates. Plates were incubated at 25±2°C for 4 to 15 d to observe conidia. Isolates had colonies and conidia consistent with members of the Botryosphaeriaceae family (Phillips et al. 2013). Immature conidia were single-celled, ellipsoidal and hyaline while mature conidia were two-celled, had a thick wall, a central septum, longitudinal striations, and a dark brown, cinnamon-like color. Size of mature conidia (n = 20) ranged 22.9 to 30.0 × 14.2 to 18.4 μm ( = 27.0 × 15.6 μm; Fig. S1). DNA templates of three isolates (23688-2_R16; 19144-18_R15 and PITA_22-1) were amplified using primers ITS1 and ITS4 for the ITS locus, EF1-688F and EF1-1251R for the translation elongation factor 1-α (TEF-1α) locus (Phillips et al. 2013) and sequenced (GenBank accession Nos. MZ413346, MZ413347, and MZ413348 for ITS; and MZ420177, MZ420178, and MZ420179 for TEF-1α). BLASTn query showed 100% identity with reference sequences of various isolates of Lasiodiplodia theobromae. Based on morphological characters and nucleotide homology, the isolates were identified as L. theobromae (Fig. S1 & S2). To fulfil Koch's postulates, 4-month-old plants of plantain hybrid PITA 24, and mature fruits from three genotypes (PITA 24, plantain cultivar Obino L'ewai) were inoculated with mycelial plugs from the margins of 5-d-old cultures of the three L. theobromae isolates. Pseudostems were drilled with a sterile 5 -mm cork borer, a mycelial plug placed down into the wound, covered with sterilized cotton, and sealed with parafilm. Sterile water was injected every third day to maintain moisture at the inoculated area. Toothpicks containing mycelia were used to inoculate fruits, placed in plastic Crisper boxes. Sterile PDA plugs or toothpicks were used for the controls. Inoculated plants and fruits were kept in a screenhouse at room temperature (~26°C) for 14 d. All inoculated materials developed symptoms similar to the diseased plants in the field. Control plants and fruits remained asymptomatic. L. theobromae was re-isolated from the artificially inoculated plant parts and its identity was confirmed. The fungus L. theobromae is distributed in tropical and subtropical regions and has a wide host range (Phillips et al. 2013; Mehl et al. 2017). This fungus was previously reported in grey literature as the causal agent of Musa spp. basal rot at Onne, Nigeria (Mwangi et al. 2005) but its molecular identification was not conducted; it was unknown whether the isolates were indeed L. theobromae or other cryptic species (L. pseudotheobromae or L. parva) (Alves et al. 2008). Over 15 years later, the present study confirms L. theobromae as the causal agent of basal rot of bananas based on nucleotide homology, and to our knowledge, this is the first report of L. theobromae causing dieback disease on plantain in Nigeria and in Africa. There is need to conduct a more comprehensive distribution surveys and develop appropriate control strategies in Nigeria.

First report of Dickeya dadantii causing bacterial soft rot of Thaumatophyllum bipinnatifidum in Taiwan.

Philodendrons are important foliage ornamentals planted worldwide (Chen et al. 2010). In November 2021, soft rot symptoms were observed on Philodendron selloum (now known as Thaumatophyllum bipinnatifidum; Sakuragui et al. 2018) grown in a nursery in Taichung, Taiwan. On symptomatic plants, the petioles were macerated; leaf lesions were also found on some plants (Figure S1). About 60% of the plants on site were symptomatic; these plants tended to cluster together. Four plants were sampled. Infected tissues were soaked and cut into pieces in 10 mM MgCl2 (using scalpels); undiluted samples were streak-plated onto nutrient agar (NA) and grown for 24 h at 28°C. Translucent, creamy-white colonies were isolated from all of the tissues examined, and 4 isolates, PHIL1 to PHIL4, were obtained (each from a different plant). All isolates exhibited typical phenotypes of bacteria belonging to Dickeya; they could cause maceration symptoms on potato slices, ferment glucose and produce phosphatase (Schaad et al. 2001); they could also produce indigoidine on NGM medium (NA added with glycerol and MnCl2; Lee and Yu. 2006). Polymerase chain reactions using Dickeya-specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon in all 4 isolates. The 16S rDNA of PHIL1 to PHIL4 were amplified using primer pair 27f/1492r (Lane 1991) and the amplicons were sequenced; all 4 isolates shared the same 1,395-bp sequence (accession nos. ON203122, ON479664-ON479666). Among the strains belonging to known species (in GenBank), PHIL1 to PHIL4 shared the highest sequence identity (99.93%) with D. dadantii 3937; they also shared 98.78% sequence identity with D. dadantii CFBP 1269T. Multilocus sequence analysis (MLSA) targeting fragments of PHIL1 to PHIL4's dnaA (720 bp), dnaJ (672 bp), dnaX (450 bp), gyrB (822 bp), and recN (762 bp) genes (Marrero et al. 2013) were conducted. The five-gene concatenated sequences (3,426 bp) of the 4 isolates (accession nos. ON227444-ON227448, ON494509-ON494523) were identical. A maximum-likelihood phylogenetic analysis including these sequences and those of type strains of other known Dickeya species revealed that PHIL1 to PHIL4 clustered with strains belonging to D. dadantii (Figure S2). Koch's postulates were fulfilled with an inoculation test conducted on T. bipinnatifidum (17 cm in aboveground height; 7-months-old). Stab inoculation using sterile toothpicks was conducted on petioles. Three plants were tested for each isolate and 2 petioles were inoculated for each plant; all 4 isolates were included in the assay. The pathogen loads inoculated were quantified by the spread plate method and were 3.22 - 4.81 x 107 colony forming units. Three plants were stabbed with bacteria-free toothpicks, serving as controls. All plants were bagged post inoculation and kept in a growth chamber (28°C; 14 h light). After 72 h, all of the inoculated petioles exhibited symptoms resembling those observed in the nursery. Bacteria were re-isolated from the symptomatic tissues (one isolate from each treatment), and all of their five-gene concatenated sequences were the same as those of PHIL1 to PHIL4. This is the first formal report of the occurrence of D. dadantii infecting T. bipinnatifidum in Taiwan. Studies have shown that D. dadantii could affect other Araceae plants in Taiwan (Lee and Chen 2021). Since different Araceae ornamentals are often planted together in gardens and nurseries, growers should be aware of potential transmission of D. dadantii among them.

First report of bacterial soft rot on Epipremnum aureum caused by Pectobacterium aroidearum in Taiwan.

Pothos (Epipremnum aureum) is an Araceae foliage plant with great ornamental values, which has long been enjoyed by consumers (Chen et al. 2010). In September 2021, pothos showing soft rot symptoms were found in 2 nurseries in Taichung, Taiwan. The petioles of the infected plants were macerated; some lesions extended to the leaves (Figure S1). The disease incidence was 50% in one nursery and 37.5% in the other; two and three plants were respectively collected from the two sites. Macerated tissues were homogenized in 10 mM MgCl2 and the samples were observed microscopically without dyeing. Motile, rod-shaped bacteria were observed in the samples, and the bacteria were isolated onto nutrient agar (NA) and grown at 28°C for 2 days. Fast-growing, round, creamy colonies were isolated from all 5 plants. One strain was isolated from each plant and the strains were named Ea1 to Ea5. The bacteria could ferment glucose and induce maceration on potato tuber slices (Schaad et al. 2001), but did not produce indigoidine on NGM medium (Lee and Yu 2006) and were tested negative for phosphatase activity (Schaad et al. 2001). The bacteria's DNA samples were tested using primers specific to Pectobacterium (Y1/Y2; Darrasse et al. 1994). The expected 434-bp amplicon was amplified in all five strains. Multilocus sequence analysis was conducted as previously described (Portier et al. 2019). A concatenated sequence (1,592 bp) comprising partial dnaX (492 bp), leuS (452 bp) and recA (648 bp) sequences was obtained for each strain. Two genotypes were detected among the strains; Ea1 and Ea2 belonged to one genotype (i.e., they had identical sequences), while Ea3, Ea4 and Ea5 belonged to the other (GenBank accession nos. OK416015-OK416020). Phylogenetic analysis was conducted using these data and those of representative strains of known Pectobacterium species (Klair et al. 2022). A maximum-likelihood tree showed that Ea1 to Ea5 clustered with P. aroidearum CFBP8168T (Figure S2). Sequence comparison (Table S1) showed that the similarity between the two genotypes' concatenated sequences was 99.1% (Ea1 vs. Ea3; 1,578/1,592 bp); Ea1 and Ea3 shared 99.2% and 99.3% sequence similarity with P. aroidearum CFBP8168T, respectively. The sequences obtained in this work were searched against GenBank and all of their top hits were those of strains belonging to P. aroidearum (supplementary information). Koch's Postulates were fulfilled by stab inoculating cutting-propagated pothos (8-cm tall) using toothpicks carrying bacteria grown on NA. The pathogen loads used were estimated by suspending cells (attached to individual toothpicks) in 10 mM MgCl2 and spread-plating them onto NA (after dilution); the loads were 5.5 x 106 - 2.2 x 107 CFU. Three plants were inoculated for each strain (3 petioles per plant). Control plants were stabbed with sterile toothpicks. Each plant was then bagged and placed in a growth chamber (28°C; 14 h light). After 24 h, all inoculated plants produced symptoms resembling those found in the nurseries, and the controls did not. For every treatment group, a strain was re-isolated onto NA; each of them shared the same recA sequence with the original strain inoculated. This is first report of P. aroidearum causing pothos soft rot in Taiwan. Local nurseries often grow pothos and other Araceae plants together in humid areas. Since other Araceae species are also known to be susceptible to P. aroidearum (Xu et al. 2020), growers should be cautious of the pathogen's spread across hosts.

First report of Lasiodiplodia theobromae causing leaf rot on Agave angustifolia in Mexico.

The agave crop (Agave angustifolia), is of economic importance for Mexico, for the agave is made mainly an alcoholic beverage called locally mezcal. In the state of Guerrero, in the municipality of Huitzuco de los Figueroa (18.2510026N, 99.2320182W, 1196 m above sea level), a severe disease affecting agave leaves was detected. The field symptoms consisted of pale to brown dark descending lesions, covering >50% of the leaf surface, in which the presence of pycnidia was observed. In an estimated area of 0.5 ha, the estimated incidence was 67% (n=100 plants). Symptomatic fragments from leaves (approximately 0.5 cm) were taken, superficially disinfected with 1% NaClO, and rinsed twice with sterile distilled water. Then they were transferred to potato dextrose agar (PDA) medium, and incubated at 28 °C. After five days, twelve representative isolates were selected and purified by the hyphal tip technique. In the PDA medium, the colonies were initially light gray, later they became dark, and after 22 days of incubation, the development of numerous dark pycnidia was observed on the surface of the medium. Initially, immature hyaline conidia, unicellular, oval, and double-walled were observed. The mature conidia were dark brown, oval, with one septum and longitudinal striation, and measured 17.5 to 27 [average 25.3 µm; n=50] × 10.5 to 15.7 [average 13.9 µm; n=50]. Based on the morphological characteristics, the fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Alves et al. 2008). Isolates LAS3 and LAS4 were used for molecular identification, this was done by amplifying the regio internal transcribed spacer (ITS) of rDNA with primers ITS1 and ITS4 (White et al. 1990) and translation elongation factor 1-alpha ( EF-1α) genes using primers EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (LAS3; ON391564 and LAS4; ON391565 for ITS, and LAS3; ON368190 and LAS4; ON368191 for EF-1α). BLASTn analysis sequences of isolated LAS3 and LAS4 revealed for ITS 98.6% identity with L. theobromae (MK934699.1), and for EF-1α indicated 100% identity (MF422024.1). From concatenated sequences ITS-EF-1α regions, a phylogenetic analysis was carried out in MEGA X software, using the Maximum Likelihood and Kimura 2-parameter model with 1,000 bootstraps replicated; isolates LAS3 and LAS4 were clustered in the clade of the members of L. theobromae strains CAA006 (Alves et al. 2006), and INTA-IMC 1601 (Perez et al. 2018). The pathogenicity tests were carried out on 10 healthy 3 year-old agave plants, in which the mycelium of the LAS4 isolate was inserted at three equidistant points/leaf, using a sterile toothpick. Five healthy agave plants were inoculated only with sterile PDA as control treatment. The inoculated plants were covered with transparent plastic bags and housed in a greenhouse at 28 °C. After seven days, similar symptoms to those observed in the field were observed in all inoculated plants. Control plants did not develop symptoms. The fungus L. theobromae was re-isolated again from the infected leaves, fulfilling Koch's postulates. In China, L. theobromae has been reported as the cause of leaf rot on A. sisalana (Xie et al. 2016). To our knowledge, this is the first report of L. theobromae causing leaf rot on A. angustifolia in Mexico. This research is useful to design management strategies for leaf rot disease for local farmers of A. angustifolia.

First Report of Neopestalotiopsis piceana Causing Gray Blight in Camellia sinensis L. in China.

Tea plants (Camellia sinensis L.) are an important cash crop and are cultivated worldwide for their commercial value (Palanisamy et al. 2014). Tea gray blight is an important tea plant disease as it can cause a decline in tea quality and reduce yields by 20-30% (Sanjay et al. 2008). In August 2018, a disease survey was conducted on 400 ha of organic tea plantations in the Pu'er area of Yunnan Province (22.48° N, 100.58° E). The survey found that widespread disease was causing damage to 40% of the tea plantations and that the most seriously affected tea variety was Yunkang No. 10, which had an average disease incidence of 30-35%. The affected leaves grew small yellow-green spots on their tips or margins in the early stage that expanded into round or irregular brown spots with distinct concentric whorls and black conidial disks arranged in whorls when the humidity was high (Fig. 1A-B), which is consistent with tea gray blight disease (Zheng et al. 2021). Twenty-four diseased leaf samples were collected from four different tea plantations and transported to the Pu-Erh Tea Research Laboratory. Leaves with disease spots were cut into 4 mm ×4 mm square pieces, surface-sterilized with 75% alcohol for 1 min, disinfected with 1% sodium hypochlorite for 3 min, and washed thrice with sterile water. The tissue pieces were placed on potato dextrose agar (PDA) plates containing 100 µg ml-1 of chloramphenicol (Wang et al. 2021). After 3 d of culturing in the dark at 28 C, twenty pure cultures with similar morphology were obtained, and two representative isolates were selected and transferred into new PDA media. After 7 d, circular fungal colonies with dense aerial mycelium produced black, wet spore masses that grew on the PDA media (Fig. 1C-D). The conidia were spindle-shaped with four septa, measuring 25.0 (21.0-26.0) × 6.0 (4.5-7.0) µm (n=15). The conidia had three median cells, two of which were dark brown in color with unclear separations, with a single basal hyaline appendage 3.8 (3.5-4.5) µm (n=30) in length and 2-3 apical hyaline appendages 31 (27-35) µm in length (n=30) (Fig. 1E), similar to the conidial characteristics of Neopestalotiopsis piceana (Maharachchikumbura et al. 2014). Two isolates were selected for DNA extraction. The internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha (tef1-α) gene, and partial β-tubulin (tub2) gene were amplified using the ITS1F-ITS4 primer set (White et al 1990), the EF-1α-F and EF-1α-R primer sets (Li et al. 2018), and the tub1 and tub2 primers, respectively (Chauhan et al. 2007). The ITS (OP535632 to OP535632), tef1-α (OP589285,OP589287), and tub2 (OP589286,OP589288) sequences were submitted to NCBI GenBank. Basic Local Alignment Search Tool analysis demonstrated that these sequences were 100% similar to those of N. piceana isolates available in GenBank. The sequences were compared using the Mafft software package, and sequences with the same ID were concatenated using scripts. A maximum likelihood phylogenetic tree was constructed using the MEGA (ver. 5.1) software package based on the concatenated sequences (ITS, tef1-α, and tub2). Phylogenetic analysis revealed that C-5 and B-3 showed 95% bootstrap support with N. piceana isolates in references (Fig. 2). According to the morphology and molecular characterization, C-5 and B-3 were identified as N. piceana. Pathogenicity tests on these two isolates were conducted using 36 healthy tea plants. The leaves were scratched slightly with sterile toothpick tips, after which pathogen cakes (6 mm diameter) were placed on the wounds with the mycelial side facing down and covered with sterile absorbent cotton to maintain a moist environment. Control leaves were wounded and covered with sterile PDA plugs (three replicates per treatment, three plants per replicate). Seven days later, the inoculated leaves exhibited similar symptoms observed under natural conditions, whereas the control leaves exhibited no symptoms. The same isolates as the introduced strains were isolated from the diseased tea leaves, completing Koch's postulates. To our knowledge, this is the first report of N. piceana causing gray blight on tea leaves in China. These results provide valuable information for the prevention and management of gray blight on tea leaves.

Isolation and Phenotypic Characterization of Lytic Bacteriophage with Activity against Pseudomonas aeruginosa

Aims: To perform the isolation and phenotypic characterization of bacteriophage with lytic activity against Pseudomonas aeruginosa. To demonstrate that this type of viral agent can be isolated from the environment and used for the biocontrol of resistant bacterial types, such as Pseudomonas aeruginosa.
 Study Design: This study was an experimental study.
 Place and Duration of Study: The study was conducted at, Bacteriology and Mycology Laboratory in the Veterinary Hospital at the School of Agricultural Sciences, Innovation and Business of the University of Passo Fundo (ESAN/UPF) and Center for Diagnosis and Research in Animal Health of the University of Passo Fundo (CDSA/UPF), between April 2022 and June 2022.
 Methodology: Samples of untreated water were inoculated with the host bacterium strain Pseudomonas aeruginosa ATCC 27853 in an enriched media After the incubation period in, a phage filtrate was obtained by centrifugation followed by filtration. We verified the presence of bacteriophages using spot test and we carried out its purification by the method of sterile toothpick plate transfer on bacterial overlay semi-solid agar. Amplification was performed using an SM buffer elution procedure to produce a stock of viral material. Through assays in Petri dishes with bacterial overlay, we performed titration and phenotypic characterization regarding the lysis spectrum and efficiency of phage infection in the host.
 Results: We managed to isolate a morphologically characterized lytic bacteriophage with approximately 1 mm of diameter, high clarity in the inhibition area, the presence of halo and well-demarcated edges. The bacteriophage, named as Pseudomonas aeruginosa Phage UPF_PaBP1, demonstrated the infection capacity of the target bacteria in all tested dilutions and a stock preparation with a titre of 6.5 x 10⁸ PFU/ml was obtained for future use.
 Conclusion: The isolated phage showed strong lytic activity against the bacterial host, a finding that nourishes our expectations regarding the use of this phage as a biocontrol agent and phage therapy.