To the authors' knowledge, this is the first report of P. ramorum occurring on C. siliqua globally and the first confirmation of Koch’s postulates for this host-pathogen combination.
Abstract
Ceratonia siliqua (Fabaceae; carob) is a woody plant native to the Palearctic and widely planted as an ornamental worldwide. In February 2024, large, irregularly shaped black necrotic lesions were detected along the leaf margins of a mature carob tree in Larkspur, Marin Co., CA (37.94779 ºN, 122.54812 ºW). Symptomatic leaves tested positive using a genus-specific lateral flow assay (LFA) for Phytophthora (Agdia Inc, Elkhart, IN, USA). Tissue segments were surface sterilized using 70% Ethanol, plated on selective PARPH media (Jeffers et al. 1986) and incubated at 20ºC. A single culture (NORS075) was isolated from the leaves, which produced morphological structures typical of P. ramorum, including coralloid mycelium with distinct hyphal swellings, chlamydospores, and semi-papillate sporangia (Fig.1) (Werres, S., et al. 2001). The isolate was identified by sequencing the internal transcribed spacer (ITS) and beta tubulin regions using the primers ITS1/ITS4 (White et al. 1990; GenBank Accession No. PX564765.1) and TUBUF2/TUBUR1 (Kroon et al. 2004; GenBank Accession No. PX614639), respectively. A BLAST search revealed 99.50% identity for ITS (796/800 bp) and 99.77% for the beta tubulin (878/880 bp) with P. ramorum ex-type strain CBS 101553 (accession no. NR_147877.1 and LC595884.1, respectively). Detached leaves of C. siliqua were inoculated with strain NORS075 using two methods: i) mycelial plugs and ii) zoospore solution (1.13 x 105 zoospores/ml). Ten unwounded and ten wounded leaves were inoculated for each inoculation method. All four treatments resulted in necrotic leaf spots (Fig.2), and P. ramorum was successfully re-isolated. Sporulation was observed on the inoculated leaves. Pathogenicity tests were also performed on whole plants (N = 20; 12cm tall and 8cm wide) of C. siliqua using two methods: i) a zoospore solution (1.9 × 105 zoospores/mL) was sprayed on ten plants until runoff; ii) leaf tips of ten other plants were immersed in 0.75 mL of the zoospore solution in a 1.5 mL microcentrifuge tube attached to the leaves. For both experiments, five leaves per plant were wounded, while the remaining leaves were left unwounded. Control plants were treated as described above but with water. Leaves inoculated by any of the four different treatments developed symptoms 5 dpi consisting of small black necrotic spots and lesions around the inoculated area (Fig.2). Symptomatic leaves tested positive using the same Phytophthora LFA, and P. ramorum was re-isolated from them. The identity of the isolates was confirmed morphologically and by sequencing the ITS region. No symptoms were detected on the control plants. To our knowledge, this is the first report of P. ramorum occurring on C. siliqua globally and the first confirmation of Koch’s postulates for this host-pathogen combination. Previously P. ramorum has been isolated from other members of the Fabaceae, such as Pickeringia montana and Cercis chinensis (APHIS 2022). Later in 2024, symptoms associated with the presence of P. ramorum were detected on approximately 90% of all ornamental carob trees in Larkspur and the neighboring town of Mill Valley, but not in other areas within Marin Co. During 2025, symptoms were detected in the same areas, but at much lower rates. The exceptionally rainy winter of 2023–2024 in the area likely contributed to high inoculum pressure and favorable infection conditions not typically found in the open, drier areas characteristic of carob habitats. Nurseries growing carob trees should survey them regularly to avoid spreading the disease to new areas.
In February 2024, common sage plants (Salvia officinalis) in a commercial greenhouse in Randolph County, North Carolina were submitted to the NC State University Plant Disease and Insect Clinic. Sage plants in multiple trays were observed to have symptoms of wilting, chlorosis, and root rot. Plants had necrotic roots and necrotic areas on leaves, and eventually entire plants collapsed. Symptomatic root segments were rinsed in sterile deionized water, blotted dry, and placed on CMA-PPP selective medium (Eckert and Tsao 1962). Three isolates, each from a different symptomatic plant, were transferred to acidified potato dextrose agar. The morphological structures from each isolate matched the description of Globisporangium irregulare (formally Pythium irregulare) reported by van der Plaats-Niterink (1981). DNA samples were extracted from mycelium mats of the three isolates using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD). The cytochrome oxidase subunits I and II (COI and COII) (Martin 2000; Robideau et al. 2011) and internal transcribed spacer (ITS) regions 1 and 4 (White et al. 1990) were amplified. The amplicons were sequenced and analyzed by BLASTn in NCBI GenBank. The isolates were confirmed to be Globisporangium irregulare with ≥98% identity match and 100% query coverage to type isolate sequences with the following accession numbers HQ708678, GU071760, and HQ643595 for COI, COII, and ITS, respectively. Isolates from this study were deposited in GenBank under accession numbers PP965484–PP965486, PP965487–PP965489, and PP965682–PP965684. To complete Koch’s postulates, ten 14-day-old sage plants were inoculated with 20 mL of 1 × 10
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oospores/mL of G. irregulare (isolate NC_65624), following a modified protocol (McGehee and Raudales 2021). Individual plants were grown in 5.7 × 5.7 × 6-cm cells cut from six-cell tray inserts filled with a soilless peat-perlite substrate. Plants were maintained indoors on growth carts at 26°C, 50% relative humidity, and a 14-h photoperiod. Wilting occurred 5 days after inoculation, and 60%–70% of inoculated plants showed reduced growth, wilting, and root rot after 14 days. Ten non-inoculated control plants remained symptomless. G. irregulare was recovered from symptomatic roots from all inoculated plants using the method mentioned previously. The experiment was conducted twice. To my knowledge, this is the first report of G. irregulare infecting common sage. G. irregulare has been reported on other herbs such as cilantro (Infante et al. 2018) and clary sage (Reeves et al. 2021). Recognizing G. irregulare as a pathogen of common sage is important for effective disease management and reducing losses in the greenhouse. Preventing root rot caused by oomycetes in greenhouse production systems requires consistent sanitation protocols, avoidance of over-fertilization and over-irrigation, and responsible use of fungicides to minimize disease risk.
Opuntia robusta is a cactus species cultivated for both fodder and fruit production. In November 2024, cladode spot symptoms were observed on approximately 60% of O. robusta plants cultivated at an experimental farm in Bloemfontein (29.1035° E, 26.1740° S), South Africa. Symptoms were characterized by light brown circular spots, with alternating rings around the areoles that coalesced to form a scab (Fig. 1A). The infection was confined to the epidermal layer. Ten symptomatic cladodes were randomly collected, surface disinfested, washed three times with sterile distilled water, and lesions (5mm2) were plated on potato dextrose agar and incubated at 28°C for 5 days in the dark. Fungal isolate HF1 had the highest isolation frequency, and was chosen for further study. The septate mycelium of HF1 was fluffy gray to black and produced branching septate conidiophores and chained conidia (19.8µm long and 10µm wide) with both longitudinal and transverse septa, consistent with the description of Alternaria sp. (Lawrence et al. 2016; Fig. 2A-D). For confirmation, genomic DNA was extracted from the isolate. The internal transcribed spacer region (ITS) region and three protein-coding genes - translation elongation factor 1-alpha (TEF 1-α), RNA polymerase II second largest subunit gene (RPB2) and Alternaria major allergen (Alt a 1) (Woudenberg et al., 2014) – were amplified using primers ITS1/ITS4 (White et al., 1990), fRPB2-5F/fRPB2-7cR (Liu et al., 1999), (O'Donnell et al., 1998) and A21F/ A21R (Gabriel et al., 2015), respectively. Sequences were deposited in GenBank under accession numbers ITS (PX112669), RPB2 (PX136066), Alt a1 (PX136067), and TEF 1-α (PX136068). BLASTn searches showed high sequence identity to Alternaria alternata with alignments of 99.8%, 100%, 99.9%, and 97.2% to ITS (MT453271.1), TEF 1-α (PV963120.1), RPB2 (PP783614.1), and Alt a1 (XM_018532577.1), respectively. In the multilocus phylogenetic tree constructed using maximum likelihood (ML) analysis of concatenated ITS-TEF1-RPB2-ALT a1 sequences, isolate HF1 clustered with reference A. alternata strains with 95% bootstrap support (Fig. 3). For pathogenicity testing, sterile toothpicks pre-colonized with isolate HF1 mycelium were used to inoculate 1-year-old O. robusta cladodes. Four inoculation points per cladode were made, with sterile toothpicks as controls. Each treatment was replicated 8 times in each of two independent trials. The cladodes were incubated in the glasshouse under natural light conditions at 25°C and 45% relative humidity and watered daily. In the first trial, 5 weeks after inoculation, 100% of inoculated cladodes had developed lesions averaging 16.4 mm in diameter, whereas no lesions were observed on the controls (Fig. 4). The second trial showed the same response pattern. In both trials, symptoms observed were consistent with those recorded under field conditions (Fig. 5). Fluffy gray to black mycelium fungi was isolated from the artificially inoculated cladodes and was confirmed as A. alternata using morphological and molecular methods as previously described, fulfilling Koch’s postulates. To our knowledge, this is the first report of A. alternata causing cladode spot on O. robusta worldwide. This finding expands the known host range of A. alternata and provides a basis for early disease diagnosis and the implementation of management strategies, including sanitation and timely fungicide applications, to reduce disease spread and protect cladodes.
Habofanoe Fosa, W. Swart, N. Muzhinji et al.· Plant Disease· 0 citations
The findings provide a theoretical foundation for accurate disease diagnosis, epidemiological surveillance, and the development of effective chemical control strategies for rubber plantations.
Zhiying Cai, Lili He, Li-Ming Dai et al.· Plants· 0 citations
In September 2025, a total of 15 soybean fields were surveyed in Muan and Gongju, Korea. Irregular leaf blight symptoms were observed in five fields, with approximately 1–10% of soybean plants showing symptoms in each affected field. To isolate the causal pathogen, symptomatic leaf tissue was surface-disinfected with 1% NaOCl for 1 min, rinsed with sterile distilled water, and placed on potato dextrose agar (PDA). The plates were incubated at 20℃ for 7 days, and olive-brown hyphae growing from the tissues were subcultured twice on PDA. Five isolates with similar morphology were obtained from symptomatic leaves, and one representative isolate, DA17-37, was selected for further morphological, molecular, and pathogenicity analyses. The conidia were predominantly oval to ellipsoidal and aseptate, measuring 3.71-6.45 x 1.77-2.83 μm, with a mean size of 5.11 x 2.14 μm (n=30). The ascospores were mostly oval and 1-septate, measuring 12.49-18.25 x 4.13-5.41 μm with a mean size of 15.08 x 4.48 μm (n=30). Additionally, multicellular chlamydospores were observed. Overall, these morphological characteristics were consistent with those of the genus Didymella. To genetically characterize isolate DA17-37, genomic DNA was extracted, and the internal transcribed spacer (ITS), beta-tubulin (tub2), and RNA polymerase II second largest subunit (rpb2) genes were amplified by PCR using the respective primer sets described previously (Gorny et al. 2016; Liu et al. 1999; Woudenberg et al. 2009). The resulting sequences were deposited in GenBank under accession numbers PZ274701, PZ433240, and PZ439292. Maximum likelihood phylogenetic analysis of the concatenated dataset showed that isolate DA17-37 clustered with the reference strains of Didymella americana. To confirm the pathogenicity of isolate DA17-37, a conidial suspension (1 x 10
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conidia/mL) prepared from 30-day-old PDA cultures grown at 20℃ was sprayed onto 2-week-old potted soybean plants. Control plants were sprayed with sterile distilled water. The inoculated plants were incubated in a dew chamber at 26℃ for 3 days and then transferred to a growth chamber maintained at 26℃ and 70% relative humidity under a 16-h photoperiod. Symptoms first appeared at 7 days post-inoculation (dpi), and representative symptoms showing disease progression were photographed at 14 dpi, whereas the uninoculated control plants remained healthy. To satisfy Koch’s postulates, the same pathogen was re-isolated from symptomatic leaves and identified based on morphology and DNA sequence analysis. Didymella americana has been reported as a causal pathogen of leaf blight on lima bean in the United States. To the best of our knowledge, this is the first report of leaf blight caused by D. americana on soybean in Korea. Continued monitoring is needed to assess the potential spread of this disease in Korea and to support the development of appropriate disease management strategies.
Soo Yeon Choi, Eun Young Kim, Shinhwa Kim et al.· Plant Disease· 0 citations
Leaf spot diseases represent a major constraint to eggplant production under favourable environmental conditions. Therefore, potential pathogenicity of the fungus Cladosporium uredinicola on the leaves of eggplant (Solanum melongena L.), cultivar 'Barcelona F1', was confirmed in this study. Symptomatic leaf tissues were obtained from several greenhouse locations in Basrah governorate, southern Iraq. The first isolation of the fungus was done on potato dextrose agar (PDA) plates, followed by the investigation of morphological and microscopic features. Cladosporium uredinicola fungal growth on the PDA medium was olive-green to dark olivaceous-brown pigmentation and irregular margins, while the microscope conidiophores were light to medium olive colorin colour, smooth to finely roughened, bearing short terminal chains of 2–5 conidia. Pale olivaceous-brown, ellipsoid to oblong in shape, smooth-walled conidia were observed with 0–3 transverse septa. The internal transcribed spacer (ITS) region was targeted for confirming the identification; the ITS sequence was deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession number PV961371.1. Pathogenicity trials were followed under greenhouse conditions, revealing the pathogenic effect of C. uredinicola on eggplant leaves, showing necrotic lesion symptoms, which initially appeared after five days post-inoculation as small brown, circular to irregular margins, subsequently extended and coalesced into large spots and patches with chlorosis symptoms and ended with leaf abscission. The fungal pathogen was re-isolated from diseased leaves to confirm Koch's postulates. The current study represents the first report of C. uredinicola as a potential pathogen on eggplant leaves in Iraq.
A. M. Angham, H. A. Mohammed· Plant Science Today· 0 citations
In March 2025, leaf spot symptoms were detected in approximately 50% of five‑month‑old Momordica charantia plants in a 1.5‑ha commercial field located in Culiacán, Sinaloa, Mexico (24°14'26.8"N, 107°11'43.2"W). Affected plants exhibited circular to irregular leaf spots lesions with grayish centers and brown to reddish margins, along with chlorosis and in advanced stages, mild defoliation. Forty symptomatic leaf samples were randomly collected for pathogen isolation. Samples were surface‑disinfected with 1% NaClO and rinsed with sterile distilled water. After drying, five tissue fragments were placed per Petri dish containing PDA supplemented with streptomycin sulfate (0.3 g L⁻¹) and incubated at 25 °C for 7 days. Among eight isolates obtained, four isolates were selected for identification: CERCCH1CULSIN, CERCCH2CULSIN, CERCCH3CULSIN, and CERCCH4CULSIN. Microscopic examination showed smooth, branched, brown and hyphae. Conidiophores were fascicles, brown, straight and unbranched, measuring 18.45–295.60 × 3.20–6.55 μm (n = 10). Conidia were hyaline, straight or slightly curved, and acicular to clavate, measuring 20.10–228.75 × 3.05–6.90 μm (n = 10). Based on these traits, the isolates were preliminarily identified as Cercospora spp. (Chupp 1954). To determine the species, the four isolates were molecularly identified by amplifying and sequencing the internal transcribed spacer (ITS) region and the translation elongation factor 1‑α (EF‑1α) using primers ITS1/ITS4 and EF1‑728F/EF1‑986R, respectively (White et al. 1990; Carbone and Kohn 1999). Sequences were deposited in GenBank under codes PQ670974, PV628715, PV628716, and PV628717 for ITS, and PQ671957, PV651779, PV651780, and PV651781 for EF‑1α, corresponding to isolates CERCCH1CULSIN, CERCCH2CULSIN, CERCCH3CULSIN, and CERCCH4CULSIN. BLAST analysis showed that ITS sequences shared about 99.8% identity with the reference C. citrullina sequence ON849061.1 (PQ670974.1, 476/477 pb; PV628715.1, 480/481 pb; PV628716.1, 479/481 pb; PV628717.1, 472/473 pb), while EF‑1α sequences showed 100% with ON890306.1 (PQ671957.1, 246/246 pb; PV651779.1, 249/249 pb; PV651780.1, 253/253 pb; PV651781.1. 253/253 pb). A Phylogenetic analysis using the Neighbor‑Joining method with 1000 bootstrap replicates grouped the isolates within C. citrullina clade. Pathogenicity of each of the four isolates was confirmed through foliar spray inoculation. Spore suspensions (1.0 × 10⁴ conidia mL⁻¹) of each isolate were applied to 15 healthy, 30‑day‑old M. charantia plants. An additional set of 15 plants was sprayed with sterile water to serve as the control. All plants were maintained at 25 ± 2 °C, 90% RH, and a 12:12 h photoperiod. Eight to ten days after inoculation, leaf spot symptoms developed and were like those observed in the field for all four isolates, whereas no symptoms were observed in the control plants. Cercospora citrullina was successfully reisolated and identified from symptomatic foliar tissue of each isolate, thereby fulfilling Koch’s postulates. This fungus has previously been reported as a causal agent of foliar disease in M. charantia (Hsieh and Goh 1990; Gautam et al. 2020); however, no prior reports exist for this pathogen on this host in Mexico. Therefore, this study constitutes the first report of C. citrullina causing leaf spot on M. charantia in Mexico, and this finding will support future research aimed at developing integrated disease management strategies for this disease.
Juan Antonio Castro-Diego, Verónica Delgado-Pacheco, N. Ley-López et al.· Plant Disease· 0 citations