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Open Research Africa 2024( ) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients...
( ) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/μL to 1×10 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.
PubMed: 38783971
DOI: 10.12688/openresafrica.14316.2 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly...
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into . After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Topics: Escherichia coli; Fructose-Bisphosphatase; Homologous Recombination; Plasmids; Genetic Vectors; DNA; Mutation; Mutagenesis, Site-Directed; Cloning, Molecular
PubMed: 38783816
DOI: 10.13345/j.cjb.230793 -
Plant Disease May 2024The fungal genus includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops,...
The fungal genus includes numerous important plant pathogenic species, some of which causes fruit bitter rot as well as leaf lesions (leaf black spot) on major crops, leading to yield losses (Fu et al. 2019; Talhinhas & Baroncelli, 2023). was reported associated with black spot on fallen, immature fruit of pear () in New Zealand (Damm et al. 2012); however, to our knowledge, this species has not been reported in Italy or nowhere else. In 2022, a significant increase of anthracnose symptoms was observed on pears in Emilia-Romagna region, Italy. Symptoms, such as round brown lesions of 1 to 4 cm, appeared on more than 50% of refrigerated stored fruit. These symptoms were undetectable at the end of September 2022 and appeared after a five-month period of storage (February 2023) at 4°C (e-Xtra 1A and B). Fungal isolates were obtained from symptomatic pears after surfaces sterilization with 96% ethanol by culturing necrotic tissue pieces on Potato Dextrose Agar at 25°C in the dark (e-Xtra 1C and D). Cultures were shades of coral color, from opalescent to sunkist coral, with slight aerial mycelium becoming grey and darker with age. When observed from the reverse side, the color was pink and, with age, became coral orange to dark amaranth. Conidia observed with a light microscope appeared hyaline and fusiform, 8 to 16 × 2.5 to 4 μm, with two pointed ends or one rounded end. (e-Xtra 1E) One reference isolate, named L51, was used for molecular characterization. Total genomic DNA was extracted, and the ITS region of rDNA amplified using the universal primers ITS1 and ITS4, then sequenced. The resulting sequences were 100% identical to those of (NR_144794.1: strain CBS 112996 ITS region; from TYPE material). Based on Damm et al. (2012), partial ACT, GAPDH, CHS and TUB2 gene sequences were also amplified and sequenced (GenBank Accession numbers: ITS: OR882016, ACT: OR882013, GAPDH: OR882011, CHS: OR882012, TUB2: OR882010), to characterize the isolates. Additionally, the multilocus phylogenetic analysis carried out with the obtained and reference sequences (Damm et al. 2012) revealed the species of analyzed isolates and confirmed the BLAST results, identifying the strain as (e-Xtra 1F). Koch's postulates were performed on 10 'Kaiser' pears. Surfaces sterilized fruits with 96% ethanol were subjected to wound inoculation with a conidial suspension (106 conidia ml-1) while 10 fruit were used as negative control and inoculated with sterile water. Following an incubation period of 8-14 days at 15-20°C, symptoms around the inoculation site resembled those initially observed, while the negative control showed no symptoms. Fungal colonies re-isolated from the lesions exhibited the same morphological characteristics as the original isolates. To our knowledge, this is the first report of pear bitter rot caused by in Italy and in Europe (Talhinhas & Baroncelli, 2023). Yet, bitter rot had not been recognized as a notable issue in pear cultivation. Nevertheless, given that pears rank as the 8th most cultivated fruit globally and economically very significant for the Emilia Romagna region in Italy the emergence of pear bitter rot caused by species has the potential to evolve into a significant worldwide problem, warranting further investigation.
PubMed: 38783583
DOI: 10.1094/PDIS-12-23-2598-PDN -
Plant Disease May 2024is a genus of trees and shrubs in the Rosaceae commonly known as rowan and mountain-ash. They are usually found in temperate regions of the Northern Hemisphere and...
is a genus of trees and shrubs in the Rosaceae commonly known as rowan and mountain-ash. They are usually found in temperate regions of the Northern Hemisphere and cultivated as ornamental trees for parks and gardens. In September 2023, infection by a rust was observed on a single tree in Sólbrekkuskógur, Reykjanesbær (64.046645, -22.707276; ~13 m) in Iceland. Infected leaves were collected from this single cultivated tree at an outdoor recreation area in a natural wooded location, with a 2% disease severity. Sori were infrequent, scattered, embedded within circular yellow lesions on leaf margins. On average, one sorus was observed per leaf and only 2% of leaves were infected. Spermogonia epiphyllous, punctate and aggregated, pale yellow to black. Hypophyllous aecia roestelioid with cornute peridium rupturing at apex with peridial cells rhomboidal, aeciospores yellowish brown globoid 17.67-25.17 x 17.20-21.94 µm, walls 1.22-2.28 µm thick (n = 20). The features of this rust and dimensions of spores are consistent with descriptions of (Arthur 1909, Kern 1911). To confirm the identity (specimen MCA9732), a ~620 bp region of the 28S subunit of the ribosomal DNA repeat was sequenced using primers Rust2inv and LR6 following published protocols (Aime 2006). The sequence (GenBank PP413765) shared 100% (649/649 bp) identity with a sequence deposited as (KY764066, BPI910184; J. E. Demers, M. K. Romberg, and L. A. Castlebury, unpublished data) from and 100% (620/620 bp) identity with (PURN11049) on sp. from Canada when blasted against the RustHUBB database (Kaishian et al. 2024). The specimen has been deposited in the Arthur Fungarium at Purdue University as PURN24233. Disease on sp. caused by has been reported in various countries in Africa, Asia, North America, and Europe (Kern 1911). To our knowledge, this is the first report of the occurrence of this genus in Iceland from any host. alternates on species. In Iceland, (sect. Oxycedrus) seems to be the only naturally occurring species but it is an alternate host for G. cornutum. The presence of the primary and alternative hosts in Iceland and the ability of spp. to produce systemic infections in spp., represents the potential for reinfection of every year, resulting in potential impacts on both host species. With being the only spp. in natural habitats in Iceland, the presence of this rust represents a potential ecological disruption, as repeated infections may reduce host vitality and predispose the host to winter injury and attack from opportunistic pathogens or insects.
PubMed: 38783582
DOI: 10.1094/PDIS-03-24-0674-PDN -
Plant Disease May 2024Microgreens are a nutrient-dense enhancement to modern diets (Choe et al. 2018), whose small production footprint in protected systems facilitates rapid crop turnover...
Microgreens are a nutrient-dense enhancement to modern diets (Choe et al. 2018), whose small production footprint in protected systems facilitates rapid crop turnover and distribution to population centers. Eleven of the 25 most broadly grown microgreens are brassicas (Choe et al. 2018). In November 2023, kale, broccoli (H009B), and cabbage (H009C) microgreen crops in Michigan were observed with downy mildew, at disease severities of 3%, 40%, and 20% foliage on 10 x 16 cm seeded blocks of plants, respectively. These crops shared a germination chamber for at least three days, which was maintained at approximately 22℃ in very humid, dark conditions. Chlorosis and grayish, sunken necrosis characterized symptoms on cotyledon surfaces (Fig. 1). In humid conditions, thick, white-light gray sporulation was present on adaxial cotyledon surfaces, accompanied by sparse sporulation on abaxial surfaces and hypocotyls. Severely diseased plants were stunted and approximately 50% gradually succumbed to downy mildew. On microscopic examination, a Hyaloperonospora spp. was tentatively identified, with long sporangiophores that dichotomously branched 3 to 6 times and hyaline sporangia borne singly on flexuous terminal sterigmata (Fig. 2). Sporangia were round to oval, with average length of 23.1 (range 16.0 to 28.3) µm; width of 20.0 (15.0 to 25.6) µm; and average length:width of 1.2 (1.0 to 1.4); (n = 97 for all). Sporangia dislodged rapidly if disturbed or as humidity decreased. Two pathogenicity tests were initiated on two sequential days. Two cotyledons from originally infected broccoli and cabbage were suspended, abaxial-side down, on coarse mesh over an open 60-mm plate of pregerminated brassica seeds on a water-saturated filter, inside a sealed, clear plastic box. Boxes contained only one type of originally diseased host, with 15 to 20 seeds of transfer varieties in unique dishes. Boxes were incubated in the dark for 2 days at 19°C with a wet paper towel atop the cotyledons. Before removal, cotyledons were lightly brushed across the surfaces of the seedlings they were just suspended above. Seedlings were grown in boxes in the presence of indirect, ambient light for 9.5 hr/day for an additional 5 days before pathogen sporulation was apparent. Filter paper was resaturated as needed. Noninoculated control plants, maintained separate from inoculated plants, were asymptomatic throughout the experiments. Total disease incidence in transfer varieties was 43.5% of 'Graffiti' cauliflower, 18.7% and 15.7% of 'Nixon' and 'Blue Vantage' cabbage; 11.8% of 'Red Russian' kale, and 6.0% of 'Ironman' broccoli, combined from two experiments. All varieties listed had at least one plant successfully infected in both pathogenicity tests. Sporulation on transfer hosts was morphologically identical to originally affected crops. Sporangiophores and sporangia were removed from H009B broccoli and H009C cabbage plants using surface sterilized forceps, placed directly into DNA extraction tubes containing buffer CD1 (Qiagen PowerSoil Pro), then kit instructions were followed. Extracts were utilized as template for ITS and cox1 PCR amplification, using DreamTaq Mastermix and ITS4/6 (45 cycles; White et al. 1990) and Levup/Levlo primers (30 cycles; Robideau et al. 2011). Cycling conditions were as published, with the number of cycles indicated by primer set. Each reaction yielded a single amplicon of approximately 1000 and 700 bp, for ITS and cox1, respectively,. Amplicons were cleaned using ExoSap-IT and submitted for Sanger sequencing, using ITS6 and Levup as sequencing primers (Robideau et al. 2011; White et al. 1990). After quality trimming, amplicons shared >98.5% identity with H. brassicae (NCBI Genbank accession MG757792 or reference genome CANTFL010000892.1). Sequences were submitted to Genbank (PP093830, PP093831, PP776812, PP776813). This is the first report of downy mildew, caused by H. brassicae, in commercial brassica microgreens, crops with vast nutritional value and expanding production.
PubMed: 38777798
DOI: 10.1094/PDIS-01-24-0266-PDN -
BMC Ecology and Evolution May 2024The ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted...
BACKGROUND
The ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted in the misidentification of species, creating challenges for understanding basic species-specific biological information that is required for restoration and aquaculture. Genetic barcoding has proven essential for accurate species identification and understanding species geographic ranges. To reduce the costs of molecular species identification we developed multiplex assays using the cytochrome c oxidase subunit I (COI or cox1) barcoding gene for the rapid identification of five species of oysters within the genus Saccostrea that are commonly found in Queensland, Australia: Saccostrea glomerata, Saccostrea lineage B, Saccostrea lineage F, Saccostrea lineage G, and Saccostrea spathulata (lineage J).
RESULTS
Multiplex assays were successful in species-specific amplification of targeted species. The practical application of these primers was tested on wild spat collected from a pilot restoration project in Moreton Bay, Queensland, with identified species (S. glomerata, lineage B and lineage G) validated by Sanger sequencing. DNA sampling by extraction of oyster pallial fluid was also tested on adult oysters collected from the Noosa estuary in Queensland to assess whether oysters were able to be identified non-destructively. DNA concentrations as low as 1 ng/ μL still amplified in most cases, allowing for identification, and mortality at 6 weeks post pallial fluid collection was low (3 out of 104 sampled oysters).
CONCLUSION
These multiplex assays will be essential tools for species identification in future studies, and we successfully demonstrate their practical application in both restoration and aquaculture contexts in Queensland. The multiplex assays developed in this study outline easily replicable methods for the development of additional species-specific primer sets for the rapid identification of other species of Saccostrea found across the Indo-Pacific, which will be instrumental in unravelling the taxonomic ambiguities within this genus in tropical regions.
Topics: Animals; Multiplex Polymerase Chain Reaction; Aquaculture; DNA Barcoding, Taxonomic; Electron Transport Complex IV; Ostreidae; Queensland; Species Specificity; Conservation of Natural Resources
PubMed: 38773413
DOI: 10.1186/s12862-024-02250-1 -
Plant Disease May 2024Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes...
Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes because of the high content of flavonoids and dihydrochalcones in the leaves (Shang et al. 2022). In July 2022, the severe leaf lesion on L. polystachyus was first observed in Yongshun County, Xiangxi autonomous prefecture (28°45'34''N, 109°40'11''E), Hunan province, China. Yongshun County is characterized by hills and mountains, situated in a subtropical region with a mild and humid climate. A second outbreak in July 2023 was observed in the same area. The observed incident rates in the past two years were 87.3% and 90.6%, respectively. Once infected, almost all plant leaves will be infected, leading to a substantial reduction in the yield of L. polystachyus. The disease presented symptoms characterized by round or irregularly shaped lesions that initially manifested as brown spots. These lesions frequently merged into larger, dark-brown areas along the leaf margins before eventually wilting. To ascertain the pathogenic species responsible for this disease, fungal isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected with 75% ethanol and 0.1% HgCl then small pieces (1×1 cm), were placed onto potato dextrose agar (PDA) medium (Sigma-Aldrich, 70139) and incubated at 28°C for 6-9 days. Colonies were villiform and initially white, becoming gray after 6 days. Sterilized dissecting needles were used to pick single hyphal tips from the edge of the colonies and placed onto PDA for strain purification. After 15 days, the purified colonies grew fluffy white hyphae with abundant conidia. The conidia were cylindrical, had round ends, and ranged from 5.75 to 14.83 μm long and 1.75 to 2.38 μm wide (n=50). According to morphological and cultural characteristics, these isolates were preliminarily identified as Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Damm et al. 2012). To further affirm the identity of the pathogen, DNA was extracted from mycelia using a DNA extraction kit (Sigma-Aldrich, G2N70). The internal transcribed spacer (ITS) region, the transcription elongation factor (TEF), and the actin (ACT) gene were then amplified from genomic DNA extracted from three isolates (Cof1, Cof2, and Cof3) using specific primers. The primers utilized were ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and ACT-512F/ACT-783R (Carbone and Kohn 1999) for ITS region, transcription elongation factor gene and actin gene amplification, respectively. Sequence identity indicated that these isolates were highly homologous to C. fructicola. The ITS (Genbank No. PP002156, OR880553 and OR880554), TEF (No. PP061421, PP061422 and PP061423), and ACT (No. PP061418, PP061419 and PP061420) sequences of the isolates Cof1, Cof2, and Cof3 shared 99 to 100% identity with their counterparts (No. OR083309, MF627961, and OQ427895) in C. fructicola, respectively. A neighbor-joining phylogenetic tree constructed using MEGA11 (Tamura et al. 2021) also indicated that these isolates were C. fructicola. Both morphological and molecular characteristics confirmed the identification of this pathogen as C. fructicola. Colletotrichum species are known to cause anthracnose disease in a variety of economically important crops (Sharma and Kulshrestha 2015). To further validate the ability of the isolated C. fructicola to induce the same symptoms as observed in the field, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspensions (1×105 conidia per mL) from three isolates were individually inoculated onto artificially wounded leaves of 3-year-old L. polystachyus. Negative controls were established by inoculating leaf wounds with sterile distilled water. The plants were incubated in a greenhouse at 28°C and 90% humidity with a 12-h photoperiod. The experiment was replicated three times. Necrotic lesions were observed on all pathogen-inoculated wounds within 6 days after inoculation, whereas controls showed no observable symptoms. Morphological and molecular characterization of re-isolated pathogens from infected leaves indicated that the pathogens were identical. To our knowledge, this is the first report of anthracnose of L. polystachyus caused by C. fructicola in China. Farmers in the local mountainous areas are economically reliant on L. polystachyus production, while anthracnose has caused over half of the trees to lose their commercial value, resulting in significant economic losses. Our findings hold great promise for advancing strategies in the prevention and treatment of anthracnose in L. polystachyus.
PubMed: 38769291
DOI: 10.1094/PDIS-03-24-0574-PDN -
PLoS Computational Biology May 2024Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to...
Recent pandemics like COVID-19 highlighted the importance of rapidly developing diagnostics to detect evolving pathogens. CRISPR-Cas technology has recently been used to develop diagnostic assays for sequence-specific recognition of DNA or RNA. These assays have similar sensitivity to the gold standard qPCR but can be deployed as easy to use and inexpensive test strips. However, the discovery of diagnostic regions of a genome flanked by conserved regions where primers can be designed requires extensive bioinformatic analyses of genome sequences. We developed the Python package krisp to aid in the discovery of primers and diagnostic sequences that differentiate groups of samples from each other, using either unaligned genome sequences or a variant call format (VCF) file as input. Krisp has been optimized to handle large datasets by using efficient algorithms that run in near linear time, use minimal RAM, and leverage parallel processing when available. The validity of krisp results has been demonstrated in the laboratory with the successful design of a CRISPR diagnostic assay to distinguish the sudden oak death pathogen Phytophthora ramorum from closely related Phytophthora species. Krisp is released open source under a permissive license with all the documentation needed to quickly design CRISPR-Cas diagnostic assays.
Topics: CRISPR-Cas Systems; Software; Humans; Whole Genome Sequencing; SARS-CoV-2; Computational Biology; COVID-19; Algorithms
PubMed: 38768250
DOI: 10.1371/journal.pcbi.1012139 -
Ecology and Evolution May 2024Islands have been used as model systems to study ecological and evolutionary processes, and they provide an ideal set-up for validating new biodiversity monitoring...
Islands have been used as model systems to study ecological and evolutionary processes, and they provide an ideal set-up for validating new biodiversity monitoring methods. The application of environmental DNA metabarcoding for monitoring marine biodiversity requires an understanding of the spatial scale of the eDNA signal, which is best tested in island systems. Here, we investigated the variation in Actinopterygii and Elasmobranchii species composition recovered from eDNA metabarcoding along a gradient of distance-to-reef in four of the five French Scattered Islands in the Western Indian Ocean. We collected surface water samples at an increasing distance from reefs (0 m, 250 m, 500 m, 750 m). We used a metabarcoding protocol based on the 'teleo' primers to target marine reef fishes and classified taxa according to their habitat types (benthic or pelagic). We investigated the effect of distance-to-reef on β diversity variation using generalised linear mixed models and estimated species-specific distance-to-reef effects using a model-based approach for community data. Environmental DNA metabarcoding analyses recovered distinct fish species compositions across the four inventoried islands and variations along the distance-to-reef gradient. The analysis of βdiversity variation showed significant taxa turnover between the eDNA samples on and away from the reefs. In agreement with a spatially localised signal from eDNA, benthic species were distributed closer to the reef than pelagic ones. Our findings demonstrate that the combination of eDNA inventories and spatial modelling can provide insights into species habitat preferences related to distance-to-reef gradients at a small scale. As such, eDNA can not only recover large compositional differences among islands but also help understand habitat selection and distribution of marine species at a finer spatial scale.
PubMed: 38766310
DOI: 10.1002/ece3.11337 -
Plant Disease May 2024Wurfbainia villosa var. villosa is a traditional Chinese herbal medicine under the family Zingiberaceae, and its ripe fruits (called Fructus Amomi) are widely used...
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.
PubMed: 38764344
DOI: 10.1094/PDIS-04-24-0717-PDN