Haixia Ding

Pseudostellaria heterophylla is a perennial herb of Caryophylaceae family, cultivated in Guizhou and Jiangsu province of China. This herb is particularly important in Chinese herbal medicine, especially valued for its reputed benefits to the immune system (Choi et al. 2016). In May 2023, the incidence of P. heterophylla leaf spot was approximately 50% in Cengong County (108° 37' E, 27° 36' N), Guizhou Province, southwestern region of China. The symptoms gradually developed from small white lesions to larger lesions. Fifteen symptomatic leaves from different plants were collected and fungal isolates were obtained by a single spore method (Liu 2010). Briefly, the conidia on the leaves were collected in a sterile tube to prepare a conidial suspension, which was gradually diluted to a single spore under a microscope and then transferred to PDA at 28°C. Finally, a total of 18 isolates were obtained. Colonies reached 89 mm in diameter after 7 days of incubation at 28°C on PDA. It was white, irregular and had gregarious conidiomata measuring 100 - 400 µm. These conidia were fusiform, slightly curved, measuring 24.5 - 35.5 × 4.0 - 5.5 µm (n=50). The basal and apical cells are hyaline, with 1, 1-3 accessory filaments, respectively, and the middle three cells are light brown. The conidiogenous cell was discrete and nearly cylindrical. Based on the morphological characteristics, the isolates were initially identified as Pestalotiopsis (Liu et al. 2017). To confirm the identification, the internal transcribed spacer region (ITS) gene, large subunit (LSU), the translation elongation factor-1 (TEF1) gene, and the β-tubulin (TUB2) gene were amplified using ITS4/ITS5, LR5/LROR, EF1-728F/EF2, and Bt2a/T1 primers, respectively (Liu et al. 2017; Carbone et al. 1999; O' Donnell et al. 1998; Santos et al. 2010). The ITS, LSU, TEF1 and TUB2 sequences of three randomly selected isolates, GUCC0218 (OR782588, OR782589, OR791708 and OR791719), GUCC0220 (PP837725, PP837723, PP855992 and PP855993) and GUCC0221 (OR782591, OR791002, OR791709 and OR791720), showed 100% nucleotide identities with the sequences of P. kenyama (CBS 442.67) in GenBank (KM199302, KM116234, KM199502 and KM199395, respectively). The phylogenetic tree based on ITS, LSU, TEF1 and TUB2 sequences, using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (Bl) methods, indicated that three isolates were most closely related to P. kenyama (CBS 442.67), supported by 100%/1/99% bootstrap values. Nine 4-month-old healthy P. heterophylla plants were sprayed with a conidial suspension (1 × 106 conidia/mL) the three isolates and three other plants were sprayed with sterile water as a control, and all treated plants were incubated in a greenhouse at 28°C (85% humidity). After 7 days, typical symptoms similar to those observed in the field appeared on the leaves of inoculated plants, while the control plants remained asymptomatic. Pestalotiopsis kenyama was re-isolated from infected leaves but not from controls. Pestalotiopsis kenyama is accountable for Camellia sinensis gray blight (Liu et al. 2017), Castanea mollissima leaf spot (Jiang 2021). Our findings will provide a basis for the correct diagnosis and management of leaf spot on P. heterophylla. To our knowledge, this is the first report of leaf spot disease caused by P. kenyama on P. heterophylla in China.

During a field study in the Baili Azalea Forest Area in Guizhou Province, China (27°12'N, 105°48'E) between May and July 2023, symptoms of leaf spot were observed on Rhododendron latoucheae Franch. The incidence of leaf spot on R. latoucheae leaves was about 12% in a field of 1 hm2, significantly reducing their ornamental and economic value. The affected leaves bore irregular, grey-white lesions with distinct dark brown borders, accompanied by black conidiomata. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. Conidiomata present on the lesions were transferred onto water agar and incubated at 25℃ for 24 hours. Germinated spores were then transferred onto potato dextrose agar (PDA) to generate single spore cultures for morphological analysis. This process yielded three single-spore isolates: GZULJ 3-6.1, GZULJ 3-6.2, and GZULJ 3-6.3, all displaying identical morphological characteristics. Representative isolate GZULJ 3-6.1 was used for further study. The colonies, cultivated on PDA at 25°C in the dark, appeared pale gray with white aerial mycelium. Alpha conidia were single celled, translucent and fusiform, measuring 6.2 to 8.1 × 2.6 to 3.0 μm (n=50). Beta conidia were filamentous and hook-shaped, measuring 17.0 to 23.0 × 0.9 to 1.5 μm (n=50). The morphological features were consistent with the established description of Diaporthe hongkongensis (Zhang et al. 2021). For molecular identification, total genomic DNA was extracted. The complete internal transcribed spacer region (ITS), partial sequence of beta-tubulin (TUB), translation elongation factor 1-alpha (TEF1), and calmodulin (CAL) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson. 1995), EF1-728F/EF1-986R and CAL-228F/CAL-737R (Carbone and Kohn. 1999), respectively. The ITS, TUB, TEF1, and CAL sequences (GenBank accession nos. OR807761, OR825453, OR825450, and OR825447, respectively) were obtained, and BLASTn analysis against sequences in GenBank showed 97.37 to 99.30% identity with Diaporthe hongkongensis CBS 115448. Phylogenetic analysis reaffirmed the isolate's placement within a well-supported cluster alongside D. hongkongensis. The pathogenicity of GZULJ 3-6.1 was evaluated using three 2-year-old R. latoucheae plants inoculated with spore suspension (106 spores per ml). The inoculation targeted three leaves per plant, while an additional three plants, serving as controls, were sprayed with sterile water. The plants were maintained in the growth chamber at 25°C, with a 12-hour photoperiod and 75% relative humidity. The pathogenicity test was repeated three times. After 14 days, the inoculated leaves developed brown lesions similar to those observed in the field, whereas the control leaves remained symptom-free. The fungus was successfully reisolated from the infected leaves and identified through morphological characterization and molecular analyses, confirming its identity. D. eres, a closely related species to D. hongkongensis, has been reported to induce similar disease symptoms on R. latoucheae (Wu et al. 2024). However, to our knowledge, this is the first report of D. hongkongensis causing leaf spot disease on R. latoucheae in China. This finding will facilitate the development of effective management strategies to control the complex leaf spot disease affecting R. latoucheae and provide critical insights into its broader implications for the health of this species.

Rosa roxburghii Tratt, known as Cili in China, is a fruit crop that grows in the mountains of southwest China at altitudes of 500 - 2500 m, especially in Guizhou province (Huang et al. 2022). In July 2021, leaf spot symptoms were observed on approximately 20 to 30% of R. roxburghii plants in a field of 6,000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou Province, China. Severe leaf spot can lead to excessive leaf drop, significantly weakening the tree and adversely affecting its growth and fruit quality, which in turn can result in reduced or even lost harvests. The symptoms appeared as irregular brown spots (0.5 to 9.5 mm), which could coalesce when densely clustered and could lead to yellowing of the leaves in severe cases. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees. Symptomatic leaves were washed with sterile distilled water and then portions of the tissue (0.5×0.5cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol, 2 min with 3% NaOCl, washed three times with sterilized distilled water), the leaves were dried and placed flat on potato dextrose agar (PDA) and left for 3-4 days incubated at 25°C (Fang, 2007). From this process, three isolates, denoted as F3-Y-21, F3-Y-22 and F3-Y-23, were obtained through single spore isolation, all displaying identical morphology. Subsequently, isolate F3-Y-21 was selected for further study. The colonies had dense aerial hyphae, initially white and later turning gray near the colony center when cultured on PDA at 28℃. Pycnidia were dark, spherical or flat spherical, and 42.2 to 52.6 μm × 51.5 to 55.2 µm in diameter (n = 50). Conidia were oval, smooth, aseptate, usually guttulate, and the size was 3.0 to 4.6 μm × 2.3 to 2.8 µm (n = 50). These morphological attributes were consistent with the description of Didymella segeticola (Chen et al. 2015). The isolate F3-Y-21 was confirmed to be D. segeticola by amplification and sequencing of the rDNA internal transcribed spacer region (ITS; primers ITS5/ITS4), large subunit ribosomal RNA gene (LSU; primers LROR/LR5), beta-tubulin gene (TUB2; primers Bt2a/Bt2b), and RNA polymerase II second largest subunit gene (RPB2; primers RPB2-5F2/fRPB2-7cR) (Liu et al. 1999; Suwannarachetal. 2019). Sequences from PCR amplification were deposited in GenBank under accessions PP159078 (ITS), PP159081 (LSU), PP178656 (TUB2), and PP178653 (RPB2). BLASTn searches of the sequences in GenBank revealed 100.00% identity of ITS (486/486 bp), 100.00% identity of LSU (574/574 bp), 98.93% identity of TUB2 (277/280 bp), and 99.05% identity of RPB2 (838/846 bp) with those sequences of D. segeticola CGMCC 3.17489 (accessions KP330443, KP330455, KP330399, and KP330414, respectively). A phylogenetic tree was constructed by MEGA7.0 using the maximum likelihood method. The isolate F3-Y-21 clustered in the same branch with D. segeticola. To assess its pathogenicity, a pot assay was conducted. Twelve leaves of three healthy R. roxburghii plants were spray-inoculated with a spore suspension (106 spores/ml), and an additional three plants were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The experiment was repeated three times. After 7 days, the inoculated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The pathogen was reisolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, LSU, TUB2 and RPB2), and the reisolated pathogen was identical to D. segeticola, thus fulfilling Koch's postulates. Similar results were obtained from three replications of the pathogenicity test. To our knowledge, this is the first report of leaf spot diseases of R. roxburghii plants caused by D. segeticola in China, although it has been previously reported to cause diseases on other hosts in China (Guo et al. 2020). It provides a theoretical basis for the detection and prevention of R. roxburghii leaf spot disease.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

Rhododendron simsii (indoor azalea) is widely cultivated for its high ornamental value (Xu et al. 2021). In April to May 2023, a leaf spot disease occurred in a field study at the Baili Azalea Forest Area (27°12'N, 105°48'E), Guizhou Province, China. About 500 plants were investigated, and the results showed that the incidence of leaf spot was 20 ~ 30%. To study this disease, 10 plants showing severe symptoms were collected. Initially, the symptoms were round or irregularly shaped brown spots (1 to 10 mm). With time, the spots enlarged and merged. Symptomatic leaves were washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were removed. After surface sterilization (30 s with 75% ethanol, 2 min with 3% NaOCl, then washed three times with sterilized distilled water), the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Fungal colonies developed from leaf tissues, and the germinated spores were transferred onto PDA for further purification and morphological observation. Three isolates (GUBJ23, GUBJ24, and GUBJ12) with similar morphology were obtained from five affected leaves. The representative strain GUBJ23 was selected for further study. On PDA the mycelium was initially white but with sporulation turned gray and then black. Black, single-celled conidia, spherical to sub-spherical, from 11.80 to 21.39 × 13.38 to 21.83 μm (n = 50) in diameter were borne singly on hyaline vesicles at the tips of conidiophores. These morphological characteristics were similar to those of Nigrospora sphaerica (Wang et al. 2017). To confirm the identification, primer pairs for the internal transcribed spacer (ITS) region (ITS5/ITS4), β-tubulin (TUB2) (Bt-2a/Bt-2b), and the translation elongation factor 1-alpha (TEF1-α) (EF1-728F/EF1-986R), were used for PCR amplification of DNA from strain GUBJ23 (Carbone and Kohn 1999; Glass et al. 1995; White et al. 1990). The resulting sequences were deposited in GenBank with accession numbers OR818025 (ITS), OR835150 (TUB2), and OR835147 (TEF1-α). BLAST searches of the sequences revealed 99.80% identity (503/504 bp) of the ITS sequence, 100.00% identity (395/395 bp) of the TUB2 sequence, and 100.00% identity of the TEF1-α sequence (241/241 bp) with N. sphaerica LC7294 (accessions KX985932, KY019602, and KY019397, respectively.) Based on a combined dataset of ITS, TEF1-α, and TUB2 sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that isolates GUBJ23, GUBJ24, and GUBJ12 were N. sphaerica (Wang et al. 2017). Leaves of three healthy R. simsii plants were spray-inoculated with a spore suspension (105 conidia/mL), and an additional three plants were sprayed with sterile water. These plants were incubated at 25℃ in 75% relative humidity. After 5 to 7 days of inoculation, 0.5 to 1.8 mm spots appeared on the leaves. At 10 to 14 days after inoculation, grayish brown, semicircular or irregular lesions appeared on the leaves, usually with a diameter of 0.8 to 3 mm. The symptoms were like symptoms seen on naturally infected leaves, while the control leaves remained asymptomatic. The pathogen was re-isolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, TUB and TEF1-α), and the reisolated pathogen was identical to N. sphaerica. Thus completing Koch's postulates. According to previous research, N. sphaerica is a widely distributed phytopathogenic fungus that has a wide host range (Wang et al. 2017). This study is the first to identify N. sphaerica as the cause of leaf spot disease in R. simsii. Given the popularity of R. simsii as a pot plant and landscape shrub in Asia and othr regions, the occurrence of leaf spot disease seriously affects its ornamental and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce impact of the disease.

Rhododendron latoucheae Franch. is an evergreen shrub with charming fragrance and large and abundant flowers that make it highly attractive as an ornamental species. The species is distribution in southwest China covers several different habitats and environments (Zhang, et al. 2022). From May to July in 2023, symptoms of leaf spot were observed on R. latoucheae over a wide portion of the Baili Azalea Forest Area (27°10' to 27°20'N, 105°04' to 106°04'E), Guizhou Province, China. About 500 plants were surveyed, and the incidence of leaf spot on R. latoucheae leaves was 12%, significantly reducing their ornamental and economic value. The affected leaves had irregular, grey white lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar, and incubated at 25°C for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GULJ1-L7, GULJ1-L8, and GULJ1-L9) identical in morphology were obtained. The isolate GULJ 1-L7 was used for further study. Colonies on PDA irregular grew white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area on the upper surface, while the lower surface presents alternating white, tan and taupe. Colony with conidiomata irregularly distributed over agar surface. In the representative isolate, darkly pigmented pycnidia (flask-shaped) were produced over the colony surface on PDA after about 30 days, and oozed milky or yellowish mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 5.15-10.29 × 1.54-3.33 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.34-31.91 × 1.01-1.90 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes and the calmodulin (CAL) using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, and CAL-228F/CAL-737R, respectively (Tao et al. 2020). Sequences from PCR amplification were deposited in GenBank with accession numbers OR740563 (ITS), OR754301 (TUB), OR754298 (TEF), and OR754295 (CAL) respectively. BLAST searches of the sequences revealed 99.07% (533/538 nt), 100% (490/490 nt), 99.69% (317/318 nt) and 98.95% (376/380 nt) homology with those of D. eres AR5193T from GenBank (KJ210529.1, KJ420799.1, KJ210550.1 and KJ434999.1), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum-likelihood method placed the isolate GULJ1-L7 in a well-supported cluster with D. eres CBS 138694T. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). The pathogenicity of GULJ1-L7 was tested through a pot assay. Due to the difficulty of artificial planting wild R. latoucheae, we conducted a pot essay to detect the pathogenicity of GULJ1-L7 using a closely related Rhododendron delavayi Franch. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying α and β spore mixture suspension (106 spores ml-1) of GULJ1-L7 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times. After 14 days, the treated leaves developed brown lesions similar to those in the field, whereas the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. latoucheae caused by D. eres in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. latoucheae.

Background: As one of the main pathogens causing tea anthracnose disease, Colletotrichum gloeosporioides has brought immeasurable impact on the sustainable development of agriculture. Given the adverse effects of chemical pesticides to the environment and human health, biological control has been a focus of the research on this pathogen. Bacillus altitudinis GS-16, which was isolated from healthy tea leaves, had exhibited strong antagonistic activity against tea anthracnose disease. Methods: The antifungal mechanism of the endophytic bacterium GS-16 against C. gloeosporioides 1-F was determined by dual-culture assays, pot experiments, cell membrane permeability, cellular contents, cell metabolism, and the activities of the key defense enzymes. Results: We investigated the possible mechanism of strain GS-16 inhibiting 1-F. In vitro, the dual-culture assays revealed that strain GS-16 had significant antagonistic activity (92.03%) against 1-F and broad-spectrum antifungal activity in all tested plant pathogens. In pot experiments, the disease index decreased to 6.12 after treatment with GS-16, indicating that strain GS-16 had a good biocontrol effect against tea anthracnose disease (89.06%). When the PE extract of GS-16 treated mycelial of 1-F, the mycelial appeared deformities, distortions, and swelling by SEM observations. Besides that, compared with the negative control, the contents of nucleic acids, protein, and total soluble sugar of GS-16 group were increased significantly, indicating that the PE extract of GS-16 could cause damage to integrity of 1-F. We also found that GS-16 obviously destroyed cellular metabolism and the normal synthesis of cellular contents. Additionally, treatment with GS-16 induced plant resistance by increasing the activities of the key defense enzymes PPO, SOD, CAT, PAL, and POD. Conclusions: We concluded that GS-16 could damage cell permeability and integrity, destroy the normal synthesis of cellular contents, and induce plant resistance, which contributed to its antagonistic activity. These findings indicated that strain GS-16 could be used as an efficient microorganism for tea anthracnose disease caused by C. gloeosporioides.