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, fulfilling Koch’s postulates.
Abstract
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.
In 2024 fall, symptoms of leaf blade and petiole spots were observed on strawberry plants 'Rociera FNM' and 'Marisma FNM'. In 450 randomly selected plants from 3 nurseries of Segovia province (Spain) 3.5% of incidence was recorded. Lesions on the leaf were reddish-brown with yellowish halos and became necrotic. Dark brown lesions with defined edges were noted on petioles. Affected tissue sections were excised, rinsed with 0.1% Tween 20, submerged in 70% ethanol (20 s), followed by 1% NaOCl (60 s), and then placed onto potato dextrose agar (Condalab, Madrid, Spain) with 50 mg/l of streptomycin sulphate. Plates were incubated at 28°C under darkness. From 6 crowns and 2 petioles 8 isolates (Di 3 to Di 10) were obtained from different pycnidia via single conidia isolation. Colonies had petal-shaped margins. Hyphae were initially hyaline, later developing an orange coloration in the center of the colony, and black conidiomata were visible two weeks post-incubation. Conidiomata were ellipsoidal to globose. Single-celled conidia were brown, ellipsoidal with slightly mucronate apices, narrowly truncated bases and smooth walls. This morphology was consistent with Coniella sp. as described by Álvarez et al. (2016). Isolates were stored in 50% glycerol solution at -80ºC. Genomic DNA was extracted from the 8 selected isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1), and large subunit ribosomal RNA (LSU) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF728/EF986 (Carbone and Kohn 1999), and LR0R/LR7 (Chethana et al. 2017), respectively. Sequence alignments performed in MEGA v11.0 (Tamura et al. 2021) showed that all isolates were identical across the three analyzed loci. Sequences of Di6 and Di10 isolates were deposited in GenBank. BLASTn analyses revealed that ITS (PX663172, PX663173), TEF1 (PX666006, PX666007), and LSU (PX663628, PX663629) sequences showed 100% identity with C. fragariae strain CBS 198.18 for ITS and 99% and 100% identity with strain CBS 167.84 for TEF1 and LSU, respectively. A concatenated phylogenetic tree was constructed with representative Coniella species and outgroup sequences from NCBI GenBank (Chethana et al., 2017) and confirmed the identity. Pathogenicity test was conducted using isolates Di6 and Di10. Inoculation with each isolate was performed by spraying 25 mL of a conidial suspension (5 x 104 conidia/mL) onto 5 plants of 'Rociera FNM'. Control plants were sprayed with sterile water. The assay was carried out twice. After inoculation, plants were covered with a plastic bag and kept in darkness for two days. Growth chamber was maintained at 25±2°C with 12-h photoperiod. Identical symptoms to the initially observed appeared on inoculated plants 6 days post-inoculation. Both isolates exhibited comparable severity (proportion of symptomatic leaf area) 20 days after inoculation. An assay was also performed on 'Marismas FNM', giving the same results. Fungal colonies re-isolated from the lesions had the same morphology and ITS, TEF-1 and LSU sequences of the inoculated isolates, thereby fulfilling Koch's postulates. In Switzerland and Great Britain C. fragariae has previously been isolated from strawberry plants (Rigotti et al. 2003; Jones and Baker, 2007). To our knowledge, this is the first report of C. fragariae on strawberry in Spain. Monitoring of the pathogen is essential since it can spread through asymptomatic plants into strawberry’s nurseries and fields.
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.
Dinh-Hieu Pham, T. Pastalka, Huiying Liu et al.· Plant Disease· 0 citations
Chestnut industry (Castanea spp.) is growing in Michigan, US; however, growers are facing emerging issues affecting the health of trees in their orchards. In July of 2025, samples from young ‘Colossal’ hybrid chestnut trees (C. sativa × C. crenata) were submitted to Plant & Pest Diagnostics from Barry County, MI. The orchard had some trees showing reddish-brown, sunken cankers on the trunk, which appeared to have expanded from the leaf node. Symptomatic woody tissues from the canker margins were surface disinfected with 10% bleach (30 s) and rinsed with sterile deionized water, then small inner sections were excised for plating on quarter-strength potato dextrose agar (PDA) amended with 40 mg/L of streptomycin and incubated in the dark at room temperature (22-24°C) until fungal colonies were observed (7-10 days). A fungal isolate exhibiting rapid growth was obtained; colonies initially produced white mycelium that darkened to gray with age and were purified by subculturing. Due to limited sporulation, the isolate was molecularly identified. After DNA extraction using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany) and amplification, sequences for three loci were obtained: internal transcribed spacer (ITS) region, β-tubulin (TUB2) and elongation factor 1-α (EF1) using the sets of primers ITS5-ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R respectively (Slippers et al. 2003; White et al. 1990). The PCR products were analyzed with NCBI BLAST. Results showed 100% identity with the ex-epitype strain Diplodia seriata CBS 112555 reference sequences for ITS (AY259094.2), Bt (DQ458856) and EF1 (Y573220). Our isolate sequences were uploaded on GenBank (NCBI) for ITS (PZ416572), Bt (PZ426090) and EF1 (PZ426091). Pathogenicity was tested on 10-month-old ‘Colossal’ seedlings using 7-day-old mycelium plugs (5 mm). The trunk of each seedling (n = 12) was surface-sterilized with 70% ethanol and wounded with razor blades about 20 cm from the soil to create a pocket across the length of the stem (2 cm). Plugs of D. seriata were inserted, and the wound was wrapped with Parafilm®. Control seedlings (n = 12) were inoculated using sterile PDA plugs. Seedlings were maintained in the greenhouse at 25/20oC day/night under a 14-h photoperiod and checked weekly for 3 months. Symptoms began developing within 21 days post inoculation. Nine of the twelve inoculated trunks started displaying dark brown stem discoloration after one month, while the untreated controls remained healthy. The pathogen was re-isolated from 9 of 12 trees using PDA. Fungal colonies morphologically identical to the original isolate were obtained. Diplodia spp. are known to cause shoot blight and canker of pines and other conifers throughout the world and recently in the USA (Blodgett et al. 2021; McKee et al. 2025).This is the first report of D. seriata causing cankers in hybrid chestnut trees in 44 Michigan (USA). This finding expands the known host range of D. seriata by documenting its association with canker disease of hybrid chestnut and highlights the need for continued monitoring of chestnut orchards and nurseries.
G. Bastianelli, A. Adaskaveg, M. Mandujano et al.· Plant Disease· 0 citations
Based on morphology and multilocus analyses, the pathogen was identified as C. eragrostidis, and the obtained sequences were deposited in GenBank placed the three isolates within the C. eragrostidis clade.
Xiang Lu, Chumiao Chen, Yunzhi Bai et al.· Plant Disease· 0 citations
Perilla frutescens (L.) Britt., an annual Lamiaceae herb, possess high medicinal, healthcare, and industrial value (Wang et al. 2020). Severe basal stem rot was recently observed on cultivated P. frutescens in Shaowu (27.34°N, 117.49°E), Fujian province, China, causing mortality ranging from 30% to 100%. Early symptoms included dark brown to black discoloration on the basal stem epidermis; mid-late stages showed brown to black discoloration of vascular bundles, followed by foliar wilting and extensive plant death. Eleven symptomatic stems were sampled, and 32 lesion marginal segments (5 × 5 mm) were obtained. Segments were surface-sterilized with 5% sodium hypochlorite for 1 min, rinsed thrice with sterile water, and air-dried. All tissues were cultured on PDA at 25°C in the dark for 7 days. Isolates were purified via single-spore separation and reincubated under identical conditions. A total of 36 isolates were obtained, including 25 Lasiodiplodia-like isolates produced compact colonies with dense aerial mycelia that changed from white/grayish-white to dark gray on PDA, and formed hyaline, ellipsoidal, unicellular conidia (22.3–27.4 μm × 12.2–15.6 μm, n = 30) on OMA (Guo et al. 2020; Wang et al. 2024). The 11 Diaporthe-like isolates developed white, fluffy aerial mycelia that grayed with age, with off-white to gray colony reverses and dark gray or pale brown centers, and yielded aseptate, smooth, ellipsoidal to clavate, biguttulate alpha conidia (5.3–7.2 μm × 1.3–3.1 μm, n = 30) on Oat Meal Agar (Zhou et al. 2025). The isolation frequency of Lasiodiplodia and Diaporthe were 69.4% and 30.6%, respectively. Genomic DNA of representative isolates (1401-10, Lasiodiplodia; 1411-11, Diaporthe) was extracted via CTAB (Stewart & Via, 1993). Partial internal transcribed spacer (ITS) (White et al. 1990), translation elongation factor (TEF-1α) (Carbone & Kohn, 1999), and beta-tubulin (TUB2) (Glass & Donaldson, 1995) sequences were amplified, with GenBank accessions PV639174/PZ287758, PZ280354/PZ295460, and PZ280355/PZ295461, respectively. Isolate 1401-10 showed 100% similarity to Lasiodiplodia pseudotheobromae ITS (MH057185), TEF-1α (OR760715), and TUB2 (KX034522), isolate 1411-11 showed 100%, 99.64%, and 99.36% similarity to Diaporthe unshiuensis ITS (MT043829), TEF-1α (KJ623300), and TUB2 (MK691289), respectively. A maximum likelihood phylogenetic tree (combined ITS, TEF-1α, and TUB2; MEGA6) clustered 1401-10 with L. pseudotheobromae and 1411-11 with D. unshiuensis (bootstrap value = 98%, Chen et al. 2021; Guo et al. 2020). Perilla seedlings were inoculated with 1% (v/v) wheat grain inoculum according to Zhong et al. 2015. Seedlings treated had pathogen-free wheat grain were served as control. The assay consisted of three biological replicates with five plants each, and the experiment repeated twice. Seedlings were covered with plastic film for 24 h and incubated at 25°C under a 12 h light/dark cycle. Ten days later, inoculated stems showed typical field symptoms, while controls remained asymptomatic. The same fungi were re-isolated from diseased stems, confirming pathogenicity. To our knowledge, this is the first report of these two pathogens causing Perilla basal stem rot in China. As a major P. frutescens producer, China faces serious threats from the emerging disease, effective management strategies are needed given their broad host ranges.
Ling Wang, Shan Zhong, Bin Wang et al.· Plant Disease· 0 citations
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).
Juan Antonio Castro-Diego, Verónica Delgado-Pacheco, N. Ley-López et al.· Plant Disease· 0 citations
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