The detailed symptoms observed after inoculation on detached healthy P. zhennan plants showing typical symptoms were similar to those of species in the genus Neopestalotiopsis, and BLAST analysis showed 93.55%–99.32% similarity with the species N. chiangmaiensis.
Abstract
Phoebe zhennan is one of the main cultivated tree species on Longquan Mountain. P. zhennan is a precious timber species endemic to China with significant economic importance. Its fine-grained, durable, and fragrant wood is highly prized for high-end furniture, traditional architecture, and handicrafts, commanding premium market prices. The species also supports rural economic development through timber production, seedling cultivation, and understory forestry industries. Additionally, it has ornamental value for landscape gardening. May 8, 2025, leaf lesions were found on 20% of P. zhennan trees within the approximately 45.5-ha survey area (Location: 30°35′50.25″N, 104°22′58.76″E, altitude 680 m). Black fruiting bodies, semi-immersed, solitary, and scattered, were produced on the diseased leaf parts. A total of 80 diseased leaves were collected from 20 P. zhennan plants showing typical symptoms. Five fungal isolates were obtained by single-conidium isolation and cultured on PDA. After 7 days of growth on potato dextrose agar (PDA) at 25 °C, the colony diameter reached 50 mm. After 30 days, fruiting bodies were produced, which were black, solitary or clustered, spherical, and released black conidial masses. Conidiophores were inconspicuous and reduced to conidiogenous cells. Conidiogenous cells were cylindrical, hyaline to pale olive. Conidia were fusiform to elliptical, mostly straight, occasionally slightly curved, with 4 septa, measuring 19–27 × 6–8 μm (x̄ = 23 ± 2.1 × 7 ± 0.4 μm, n = 50); the basal cell was conical to obconical, hyaline, 3–5 μm long (x̄ = 4 ± 0.7 μm, n = 50); the three middle cells were polygonal to cylindrical, 13–19 μm long (x̄ = 15 ± 1.5 μm, n = 50), slightly constricted at the septa, pale olive to dark brown, with the central cell darker and the flanking cells slightly lighter, thick-walled, wrinkled, and dark brown to black; the apical cell was hyaline, conical to trapezoidal, thin-walled, and smooth; it possessed 2–4 tubular apical appendages, unbranched, filamentous, hyaline, 18–29 μm long (x̄ = 23 ± 2.9 μm, n = 50); the basal appendage was solitary, tubular, straight, occasionally slightly curved, hyaline, 2–4 μm long (x̄ = 3 ± 0.5 μm, n = 50). The detailed symptoms observed after inoculation on detached healthy P. zhennan leaves with mycelial plugs of N. chiangmaiensis were recorded. The morphological characteristics were similar to those of species in the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). Two representative isolates (TXC202505-1 and TXC202505-2) were selected for DNA extraction, and the ITS, tef1-α, and tub2 gene regions were amplified and sequenced (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999; Maharachchikumbura et al. 2014). All sequences have been deposited in GenBank (ITS: PX930896–PX930897; tub2: PX938982–PX938983; tef1-α: PX938978–PX938979). BLAST analysis showed that these sequences shared 93.55%–99.32% similarity with the species N. chiangmaiensis (type strain MFLUCC 18-0113): ITS: 174/186 bp (93.55% and 93.55%); tef1-α: 925/927 bp (99.78% and 99.78%); tub2: 438/441 bp (99.32% and 99.32%). Based on multi-gene phylogenetic analysis, the isolates clustered together with N. chiangmaiensis with a support rate of 72%. Morphologically, the isolated strains were consistent with N. chiangmaiensis, and their nucleotide sequences exhibited high similarity. Integrating morphological and molecular phylogenetic evidence, the isolates were identified as N. chiangmaiensis. To confirm pathogenicity, three healthy two-year-old seedlings were inoculated with 500 μl of a spore suspension (1 × 10⁵ conidia/ml) on five leaves each. Three control seedlings were inoculated with sterile distilled water. All treated leaves were bagged and maintained in a greenhouse at 25°C and 70% relative humidity. The test was repeated three times. The detailed symptoms observed after inoculation are as follows: Initial symptoms appeared as small, water-soaked, light brown spots at the inoculation sites 3–5 days after inoculation. The spots gradually expanded into circular or irregular necrotic lesions with dark brown margins and grayish-white centers. Under high humidity conditions, black acervuli (asexual fruiting bodies) developed on the lesion surfaces. In severe cases, the lesions coalesced, leading to large areas of leaf necrosis and blight. Control leaves inoculated with sterile water remained asymptomatic throughout the experiment. After ten days, symptoms similar to those observed in the field developed on the inoculated leaves, while no symptoms were observed on the control plants. N. chiangmaiensis was reisolated from symptomatic leaves (100% frequency) and identified based on morphological and molecular characters, fulfilling Koch’s postulates. N. chiangmaiensis has been previously reported on plants such as orchids, roses, and nuts (Guterres et al. 2023; Sun et al. 2023). N. chiangmaiensis was originally described from Thailand and has also been reported in southern China as a saprobe on Pandanaceae plants (Tibpromma et al. 2018). However, this is the first report of N. chiangmaiensis causing leaf spot disease on P. zhennan, representing a new host record in China. This is the first report of this fungus causing leaf blight on P. zhennan in Longquan Mountain, Sichuan Province.
Conioselinum anthriscoides is a perennial herb belonging to the Apiaceae family. It grows wild on mountain slopes and forest edges, primarily at elevations between 1,300 to 1,800 meters. It is mainly cultivated in Sichuan, Shaanxi, Shandong and Xinjiang provinces. Its rhizomes are used in traditional medicine for dispelling wind and relieving pain (Committee of China Pharmacopoeia, 2020). In June 2019, severe leaf spot disease was observed on the leaves of C. anthriscoides in a 150 m2 field at a medicinal plant garden in Min County, Gansu Province, China (35°17’N, 104°2’E, altitude 2489.3 m). Initial symptoms on infected leaves appeared as small, irregular, brown necrotic spots. These lesions gradually turned dark brown to grayish-white, developing small black dots and exhibiting pronounced dark brown margins. Disease incidence was estimated at 60 to 75% across the field based on a 5-point random sampling survey of 300 leaves. The pathogen was obtained using the tissue isolation method on potato dextrose agar (PDA) at 25°C, while no fungus was isolated from asymptomatic leaves processed similarly. Isolate TCM-22, obtained with an isolation frequency of 53%, was selected as the representative isolate for further analyses.The colonies on PDA exhibit well-defined margins, dense aerial mycelium initially appears white, becomes grayish-olive with age. Pycnidia were black, spherical to subspherical, produced on PDA after 3 weeks, measuring 52.8 to 112.7 × 43.5 to 107.5 (avg. 84.8 × 75.9) μm , with ostioles 25.1 to 35.8 × 22.7 to 33.6 (avg. 32.4 × 28.9) μm in diameter. Conidia were hyaline, acicular (needle-like), straight or slightly curved, with 2 to 4 septa, measuring 18.3 to 52.8 × 1.4 to 3.8 (avg. 35.7 × 2.7) μm. These morphological characteristics were consistent with the genus Septoria. S. anthrisci TCM-22 exhibits mycelial growth at temperatures ranging from 4 to 30°C, with an optimum at 20°C. Continuous illumination enhanced growth relative to darkness. The fungus grows at pH 5.0 to 10.0, with optimal growth at pH 5.0. After culturing isolate TCM-22 on PDA medium for 20 days, 10 mL of sterile 0.1% Tween-80 solution was added to each petri dish, and the fungal culture was scraped with a spatula to prepare a spore suspension. Adjust the suspension concentration to 1 × 105 spores/mL using sterile water. Thirty healthy one-year-old plants were selected for the pathogenicity test. Fifteen plants were inoculated by spraying with the spore suspension until it runs off the edges of the leaves. The remaining sprayed with sterile 0.1% Tween-80 solution were treated as control. All plants were covered with transparent plastic bags to maintain 80% relative humidity and placed at 20℃ in the Climate Chamber for 72 hours under a 12-hour photoperiod. Then the plastic bags were removed. Twenty days post-inoculation, all leaves inoculated with spore suspension exhibited dark brown necrotic lesions with black margins, resembling the original field symptoms. No symptoms were observed in the control group. The experiment was repeated three times, and the pathogen was again isolated from the inoculated leaves, with consistent results. This satisfied Koch's postulates, confirming the pathogen's pathogenicity. The DNA of isolate TCM-22 was extracted using the FastDNA kit (SK1375) (Sangon Biotech Co. Ltd. , Shanghai, China) and 3 loci: ITS, β-tubulin and EF1α were amplified and sequenced with primers ITS1/ITS4, T1/β-Sandy-R and EF1-728F/EF-2 respectively (Verkley et al. 2013). The obtained sequences of ITS, β-tubulin, EF1α were deposited in GenBank (accession nos. PQ815052, PQ827513, and PQ827514, respectively. BLAST results of the three gene loci indicated 99% to 100% sequence identity with S. anthrisci TCM-11, CBS 109020 and CBS 109020. A phylogenetic tree based on the concatenated sequences of ITS, β-tubulin, and EF1α was constructed using the Maximum Likelihood method. The strain TCM-22 formed a single clade with S. anthrisci TCM-11. Based on morphological and molecular data, the isolate was identified as S. anthrisci. To our knowledge, this is the first report of S. anthrisci causing leaf spot on C. anthriscoides in China.
Rou Sun, Yan Wang, Ling Jin et al.· Plant Disease· 0 citations
Ligustrum lucidum, commonly known as Chinese privet, is an ornamental tree widely cultivated in China. In August 2024, a severe leaf spot disease was observed on Ligustrum lucidum in and around Dushan Forest Park (33°04′13″N, 112°35′11″E) in Nanyang, China. Disease incidence reached 59% among 100 surveyed trees, reducing the ornamental value of affected plants. The lesions were initially small, scattered, and pale gray on the adaxial leaf surface, mostly circular to irregular in shape. On the abaxial surface, the corresponding lesions were slightly sunken and light brown. As the disease progressed, adjacent lesions enlarged and coalesced, and the necrotic tissue occasionally detached, producing shot-hole-like perforations. For pathogen isolation, ten symptomatic leaves were randomly collected. Small tissue segments were excised from the margin between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s, followed by 1% NaClO for 1 min, rinsed twice in sterile water, and plated on potato dextrose agar (PDA) supplemented with 50 μg/ml streptomycin. Plates were incubated at 25°C in darkness. In total, 16 isolates with similar morphological characteristics were obtained. Three representative isolates (DYNZ09, DYNZ17, and DYNZ20) sampled at separate sites were selected for further investigation. Colonies grew slowly on PDA at 25°C, with a growth rate of 3 mm per day. Conidia were light brown and obclavate to cylindrical, measuring 16.3–95.0 × 4.5–10.7 μm (n = 100). These morphological features were consistent with descriptions of Corynespora cassiicola. For molecular identification, genomic DNA of three representative isolates was extracted using the CTAB method. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α), and β-tubulin genes (tub) were amplified using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were deposited in GenBank (ITS: PX488446–PX488448; tef1-α: PX549115–PX549117; tub: PX549119–PX549121). BLAST analysis showed 99.1-100% identity with sequences of C. cassiicola. Phylogenetic analysis based on concatenated ITS, tef1-α, and tub sequences using the maximum likelihood method (MEGA12) (Kumar et al. 2024) demonstrated that the isolates clustered with C. cassiicola strains. Pathogenicity assays were performed by spraying a conidial suspension (106 conidia ml-1) onto healthy leaves of L. lucidum seedlings (60–80 cm in height). Control plants were treated with sterile water. Inoculated seedlings were maintained in chambers at 28°C and 90–95% relative humidity under a 12 h light/12 h dark photoperiod. At 14 dpi, inoculated seedlings developed foliar symptoms, which were identical to the field observations. Control plants remained symptomless. C. cassiicola was consistently reisolated from diseased tissues, thereby fulfilling Koch’s postulates. C. cassiicola is a well-known pathogen with a broad host range and has been reported to cause diseases on soybean (Lu et al. 2021). C. cassiicola has been reported on other Ligustrum species, including L. quihoui (Yu et al. 2023) and L. × vicaryi (Wang et al. 2013). However, these reports involved different host species. To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. lucidum in China. This finding provides a basis for accurate diagnosis and future management of Corynespora leaf spot on L. lucidum.
Tan Wang, Zhanying Zhu, Zi-Meng Kou et al.· Plant Disease· 0 citations
Michelia × alba (M. alba), an evergreen magnolia, is widely planted as courtyard and street ornamental tree across subtropical South China. It grows in open ground in southern provinces and pots in the Yangtze River basin (Liang and Nooteboom 1993). In February 2026, severe anthracnose was observed on M. alba in the Qujing community courtyard, Qujing City (103.783028 E, 25.472859 N), Yunnan Province, China. Disease symptoms were observed on 60 - 80% of leaves in planting plots (~ 0.015 - 0.020 ha) with 15 - 20 mature M. alba trees. The disease forms small water-soaked leaf spots expanding into irregular brown lesions with clear yellow halos. Lesions range 1.2 × 1.0–4.5 × 3.8 cm, with sunken necrotic centers and concentric rings. Under humid conditions, lesions coalesce to trigger widespread leaf blight and early leaf drop, with dark fungal fruiting bodies occasionally present on dead tissue. To isolate the fungus, 9 symptomatic leaves were randomly collected from three trees and rinsed with sterile water. Small, infected tissue segments (~ 5 mm²) were surface sterilized with 75% ethanol for 30 s, then 3% hypochlorous acid for 3 min, rinsed thrice with sterile water, and inoculated on potato dextrose agar (PDA). Plates were incubated at 28°C with a photoperiod of 12 h. 9 strains with similar morphology characterizations were isolated, and two representative isolates (BLYB1 and BLYB2) were purified. Purified colonies initially had sparse edges on PDA, becoming dense after 3 days, covering the dish by 12 days, with round, flat edges. Microscopically, the conidia were observed to be hyaline, cylindrical, and obtuse at the apex, measuring (12.30 - 16.90) × (5.56 - 8.36) μm, avg. (14.85 ± 2.30) × (6.96 ± 1.40) μm (n = 50). These morphological characteristics resembled Colletotrichum siamense. To confirm identification, genomic DNA from mycelium of these two isolates was extracted, and the sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS1), and calmodulin (CAL) were amplified (Zhang et al. 2020), with GenBank accessions: ITS (PZ537092, PZ537093), GAPDH (PZ584306, PZ584307), ACT (PZ584308, PZ584309), CHS1 (PZ584310,PZ584311), CAL (PZ584312, PZ584313). Pathogenicity of isolates BLYB1 and BLYB2 was assayed on healthy one-year-old leaves with needle wounding, using 5 mm mycelial plugs in three biological replicates. Inoculated leaves were cultured at 25 - 30℃, 85 - 95% relative humidity and a 12 h photoperiod. Typical leaf spots formed on all inoculated leaves at 5 days post-inoculation, whereas control leaves treated with blank PDA plugs or sterile water showed no lesions. The target fungus re-isolated from diseased tissues was identified as C. siamense, which fulfilled Koch’s postulates. The fungus was re-isolated and identified as C. siamense, fullfilling Koch's postulates. C. siamense infects at least 60 plant species across the world (Ji et al. 2019). To our knowledge, this is the first report of C. siamense causing leaf spot on M. alba in Yunnan, China via multilocus phylogenetic analysis. This finding aids targeted disease management to protect M. alba plantations, as C. siamense anthracnose reduces the ornamental value of M. alba, and supports the need for research on host-pathogen interactions and resistance breeding.
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
Idesia polycarpa, a deciduous tree in the Salicaceae family, is used for oil and timber (Wu et al. 2019). In August 2025, a field survey at an I. polycarpa nursery in Zunyi City, Guizhou Province (27.9° N, 106.9° E) revealed leaf disease on seedlings, with 50% disease incidence (n=1000). Early symptoms were small light yellow spots, expanding into irregular brown to black lesions with dried margins, necrotic areas, and defoliation. Lesion edges were yellowish, centers grayish-brown to blackish-brown. Twenty diseased leaves were randomly selected from five plants. Tissue pieces (5 mm × 5 mm) from lesion margins were disinfected with 3% sodium hypochlorite for 30 s and 75% alcohol for 1 min, rinsed with sterile distilled water, placed on PDA medium, and incubated at 28℃ for five days. Three morphologically similar strains, HB-1, HB-2, and HB-869, were isolated. On PDA at 28℃ in the dark, colonies were white on the obverse and light yellow on the reverse. The texture was velvety to floccose; the aerial mycelium was well-developed, dense, and fluffy, evenly covering the entire plate. Acervuli were black, spherical to subspherical, 0.15-0.97 mm in diameter, and embedded in the mycelial layer. All isolates produced asexual structures, with black setae measuring 37.7–39.5 μm × 4.7–4.9 μm, having a cylindrical base and a gradually tapering tip. Appressoria were ellipsoidal, dark brown, measuring 6.90–11.26 μm (mean = 8.14 ± 1.26 μm) × 5.08–7.94 μm (mean = 6.50 ± 0.92 μm) (n = 50). Conidiophores were colorless to light brown, septate, and branched. Conidia were slender ovoid, colorless, unicellular, aseptate, and smooth-walled, with rounded ends and containing oil globules, measuring 8.24–13.24 μm (mean = 11.50 ± 1.35 μm) × 4.51–5.18 μm (mean = 4.87 ± 0.22 μm) (n = 50). Cylindrical, rounded-end conidia were observed, matching the morphology of C. fructicola (Khuna et al. 2025). For identification, representative isolate HB-869 was subjected to multi-locus sequence analysis using ITS, CHS-1, and GAPDH, with primers ITS1/ITS4, CHS-79F/CHS-345R, and GDF/GDR, respectively (Weir et al. 2012). BLAST searches revealed that the ITS (GenBank: PX992721), CHS-1 (PZ094120), and GAPDH (PZ094121) sequences of HB-869 showed 98.89%, 100%, and 98.57% identity to those of the C. fructicola ex-type strain ICMP 18581 (JX010165, JX009866, and JX010033, respectively). Based on morphological and molecular evidence, HB-869 was identified as C. fructicola. For pathogenicity assay, ten 1-year-old I. polycarpa seedlings, approximately 40 cm tall, were selected. Leaves were inoculated by sterile needle-pricking. Five plants received 5 mL of HB-869 conidial suspension (1×10⁶ conidia/mL), while five control plants received sterile distilled water. All were incubated at 25 °C, 70% RH, under a 12 h light/dark cycle. The assay was independently repeated three times. At 14 days post-inoculation, typical anthracnose symptoms appeared on inoculated plants, consistent with field observations, while controls remained asymptomatic. The re-isolated pathogen was identified by morphology and ITS, CHS-1, and GAPDH sequences, fulfilling Koch’s postulates. C. fructicola infects diverse hosts, including Epimedium sagittatum (Hou et al. 2024). Given the economic importance of I. polycarpa, this finding expands the known host range of C. fructicola and offers valuable insights for disease diagnosis and management. This is the first report of anthracnose on I. polycarpa caused by C. fructicola in China.
Lanxin Sun, Cheng-Xu Wu, Zaihua Yang et al.· Plant Disease· 0 citations
Ligusticum chuanxiong Hort., the dried rhizome of which is a commonly used herb for its therapeutic purposes in Chinese medicine (Wang et al. 2025). Root rot is a prevalent and devastating disease in the cultivation of L. chuanxiong, with 20–50% incidence in major production areas, leading to significant yield losses. The disease occurs throughout the growing period, with the most severe damage occurring at the seedling stage. Infected plants exhibit brownish lesions inside basal stems and roots, progressive softening and water-soaking of internal tissues, and rhizome decay with a sour odor as the disease progresses. In July 2024, three symptomatic root samples of L. chuanxiong were collected from Anguo, Baoding City, Hebei Province, China (38°22′25″N, 115°21′18″E). Symptomatic roots were rinsed thoroughly with clean water, surface-disinfected by immersing in 5% sodium hypochlorite solution for 2 minutes, followed by three washes with sterile distilled water. The roots were dissected by a sterile scalpel, and tissue pieces of approximately 5 mm were excised from the junction between diseased and healthy areas. These segments were plated on potato dextrose agar (PDA) and incubated at 25°C under dark conditions for 3 days. Emerging hyphal tips growing from the disinfected tissues were transferred to fresh PDA plates, and single-spore isolates were obtained by dilution plating. Six isolates with similar colony morphology were obtained. The colony of the representative isolate initially developed white, floccose-to-cottony aerial mycelia that formed a vigorous, dense mat and pale purple pigments with prolonged incubation. To induce sporulation, the isolate was cultured on carnation leaf agar (CLA) at 25°C under a 12 h light/12 h dark regime for 7 days. Macroconidia were typically falcate, with 3 to 5 septa, and measured 22.8 to 44.9 × 3.8 to 7.2 μm (n=50), and microconidia were ovoid, with 0 to 1 septum, measuring 4.8 to 14.4 × 3.3 to 6.8 μm (n=50). These morphological characteristics are consistent with the description of Fusarium commune (Skovgaard et al. 2003). To identify these isolates, the representative isolate designated FC-1 was selected for molecular identification. Genomic DNA was extracted and three loci were amplified and sequenced: the internal transcribed spacer (ITS) region using primers ITS1/ITS4 (White et al. 1990); the translation elongation factor 1-alpha (TEF-1α) gene using primers EF1/EF2; and the RNA polymerase second largest subunit (RPB2) gene using primers RPB2-5f2/RPB2-7cr (O’Donnell et al. 2010). Forward and reverse sequences were assembled to generate consensus sequences for each locus. The consensus sequences were deposited in GenBank with accession numbers PX487973 (ITS), PX529605 (TEF-1α) and PX529607 (RPB2). BLASTn analysis revealed that the sequences exhibited 100% identity to Fusarium commune. Phylogenetic analysis of the concatenated TEF-1α and RPB2 gene sequences indicated that FC-1 clustered with F. commune. To assess pathogenicity, the isolate was cultured in 100 mL of potato dextrose broth (PDB) at 25°C with constant shaking at 200 rpm for 4 days. Cultures were filtered through 4 layers of sterile cheesecloth to obtain spore suspensions (1×106 spores/mL). Healthy L. chuanxiong plants were selected for whole-plant pathogenicity tests. After washing and surface sterilization, the roots were immersed in spore suspensions for 30 minutes and subsequently potted in sterilized substrate. Control roots were treated with sterile distilled water. The pathogenicity test was conducted with three replicate pots per treatment and repeated three times independently. All plants were incubated at 25°C with 85% relative humidity under a 12 h light/12 h dark regime in a growth chamber. After 7 days incubation, inoculated plants developed typical symptoms, including aboveground wilting and chlorosis, as well as internal rot of the rhizome, whereas control plants remained asymptomatic. Three independent assays demonstrated the consistent pathogenicity of FC-1 to L. chuanxiong. The pathogen was successfully re-isolated from symptomatic tissues and identified via sequencing as the original inoculum, thereby fulfilling Koch’s postulates. This study represents the first confirmed report of F. commune as the causal agent of root rot in L. chuanxiong, highlighting a novel and significant threat to the sustainable cultivation of this important medicinal plant.
Bing-Yan Xia, Jie-Yin Chen, X. Dai et al.· Plant Disease· 0 citations
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