Bletilla striata (Thunb.) Rchb. f. (Orchidaceae), known as a traditional Chinese herb, has been widely used as hemostatic and anti-microbial. In May 2017, a leaf spot disease was observed on 20–30% of B. striata in commercial fields located in Ziyuan County, Guangxi, China. Small, circular, brown spots were early observed on leaves, enlarging and developing into irregular dark brown and necrotic lesions. Necrotic lesions caused leaf abscission and occurred frequently during the plant’s life cycle. Three symptomatic leaves from three individual plants were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed 3 times in sterile water, plated on potato dextrose agar (PDA) medium and then incubated at 26°C for 3 days. Seven isolates recovered from diseased plants and the colonies were villose and regular, pale grey, secreting scarlet pigment to the media. After 15 days of incubation, subspheroical chlamydospores were observed, which were brown to black, with verrucose surface. Unicellular chlamydosp...

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant. In April 2018 and 2019, a leaf spot disease was observed on ∼20% of B. striata plants in two fields (∼1.4 h) in Guilin, Guangxi Province, China (Fig.1 A). Small, circular, brown spots were initially observed on the leaf surfaces, which progressively expanded into large, sunken, dark brown, necrotic areas. As the disease progressed, lesions merged into large, irregular spots, ultimately resulting in abscission. To determine the causal agent, small pieces (5 mm x 5 mm) were collected from the infected leaf tissues (n = 18), surface sterilized in 1% NaOCl for 2 min, and rinsed three times with sterile water. Then, the tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/L) and incubated under 12 h photoperiod at 26°C for 3 days. Seventeen isolates were obtained, of which twelve isolates with similar morphological characteristics were obtained from the germinated spores on PDA. Seven-day-old colonies on PDA appeared cottony, pale white to pale gray from above, and grayish-green from below. Conidia of strain BJ-101.3 were hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig.1 E-G), measuring 11.3 to 15.9 μm × 4.0 to 6.4 μm (n = 50). Appressoria were brown to dark brown, with different shapes and a smooth edge (Fig.1 H-I), measuring 6.3 to 10.0 μm × 4.1 to 8.0 μm (n = 50). Morphological features were similar to C. gloeosporioides species complex (Weir et al. 2012, Fuentes-Aragón et al. 2018). For molecular identification, DNA was extracted from two isolates BJ-101.3 and BJ-101.13, following the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese superoxide dismutase (SOD2), beta-tubulin (TUB2), glutamine synthetase (GS), and Apn2-Mat1-2 intergenic spacer and partial mating-type (ApMat) genes were amplified by PCR and sequenced (Weir et al. 2012, Silva et al. 2012, Vieira et al. 2017). The obtained sequences were deposited in GenBank (MW386818, MW386819, MW403508 to MW403519, and MW888410 to MW888413). BLASTN analysis of the obtained sequences showed 99% identity with those of C. fructicola (JX010165，JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405, JQ807838) (Weir et al. 2012, Liu et al. 2015). A phylogenetic tree based on the concatenated sequences confirmed the isolates as C. fructicola (Fig.2). Furthermore, pathogenicity tests were conducted on six 1.5-year-old B. striata plants. Healthy leaves on the plants were inoculated with the conidial suspensions (106 conidia/mL; 10 μL) of the strains BJ-101.3 and BJ101.13. The conidial suspension of each isolate was inoculated onto at least three leaves. Another three plants inoculated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C for 14 days with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained symptomless (Fig.1 B-C). The experiments repeated three times showed similar results. Finally, C. fructicola was consistently reisolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. The outcome of this study will help in developing effective management measures against anthracnose of B. striata.

Curcuma kwangsiensis S. G. Lee et C. F. Liang is a traditional Chinese medicinal plant distributed in Guangxi and Yunnan Province, China. In May 2021, a leaf blight disease on C. kwangsiensi was ob...

Banana (Musa acuminata) is one of the most important fruit crops in the world. In June 2020, a leaf spot disease was detected on M. acuminata (AAA Cavendish cv. Williams B6) in a commercial plantat...

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is traditionally used for hemostasis and detumescence in China. In April 2019, a leaf spot disease on B. striata was observed in plant nurseries in Guilin, Guangxi Province, China, with an estimated incidence of ~30%. Initial symptoms include the appearance of circular or irregular brown spots on leaf surfaces, which progressively expand into large, dark brown, necrotic areas. As lesions coalesce, large areas of the leaf die, ultimately resulting in abscission. To isolate the pathogen, representative samples exhibiting symptoms were collected, leaf tissues (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface-disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA) medium, and incubated at 28°C (12-h light-dark cycle) for 3 days. Hyphal tips from recently germinated spores were transferred to PDA to obtain pure cultures. Nine fungal isolates with similar morphological characteristics were obtained. Colonies on PDA were villose, had a dense growth of aerial mycelia and appeared pinkish white from above and greyish orange at the center and pinkish-white at the margin on the underside. Macroconidia were smooth, and hyaline, with a dorsiventral curvature, hooked to tapering apical cells, and 3- to 5-septate. Three-septate macroconidia were 21.2 to 32.1 × 2.4 to 3.9 μm (mean ± SD: 26.9 ± 2.5 × 3.2 ± 0.4 μm, n = 30); 4-septate macroconidia were 29.5 to 38.9 × 3.0 to 4.3 μm (mean ± SD: 33.5 ± 2.6 × 3.6 ± 0.3 μm, n = 40); and 5-septate macroconidia were 39.3 to 55.6 × 4.0 to 5.4 μm (mean ± SD: 48.0 ± 3.9 × 4.5 ± 0.3 μm, n = 50). These morphological characteristics were consistent with F. ipomoeae, a member of the Fusarium incarnatum-equiseti species complex (FIESC) (Wang et al. 2019). To confirm the fungal isolate's identification, the genomic DNA of the single-spore isolate BJ-22.3 was extracted using the CTAB method (Guo et al. 2000). The internal transcribed space (ITS) region of rDNA, translation elongation factor-1 alpha (TEF-1α), and partial RNA polymerase second largest subunit (RPB2) were amplified using primer pairs [ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (O'Donnell et al. 1998), and 5f2/11ar (Liu, Whelen et al. 1999, Reeb, Lutzoni et al. 2004), respectively]. The ITS (MT939248), TEF-1α (MT946880), and RPB2 (MT946881) sequences of the BJ-22.3 isolate were deposited in GenBank. BLASTN analysis of these sequences showed over 99% nucleotide sequence identity with members of the FIESC: the ITS sequence showed 99.6% identity (544/546 bp) to F. lacertarum strain NRRL 20423 (GQ505682); the TEF-1α sequence showed 99.4% similarity (673/677 bp) to F. ipomoeae strain NRRL 43637 (GQ505664); and the RPB2 sequence showed 99.6% identity (1883/1901 bp) to F. equiseti strain GZUA.1657 (MG839492). Phylogenetic analysis using concatenated sequences of ITS, TEF-1α, and RPB2 showed that BJ-22.3 clustered monophyletically with strains of F. ipomoeae. Therefore, based on morphological and molecular characteristics, the isolate BJ-22.3 was identified as F. ipomoeae. To verify the F. ipomoeae isolate's pathogenicity, nine 1.5-year-old B. striata plants were inoculated with three 5 × 5 mm mycelial discs of strain BJ-22.3 from 4-day-old PDA cultures. Additionally, three control plants were inoculated with sterile PDA discs. The experiments were replicated three times. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 26°C for 14 days. Four days post-inoculation, leaf spot symptoms appeared on the inoculated leaves, while no symptoms were observed in control plants. Finally, F. ipomoeae was consistently re-isolated from leaf lesions from the infected plants. To our knowledge, this is the first report of F. ipomoeae causing leaf spot disease on B. striata in China. The spread of this disease might pose a serious threat to the production of B. striata. Growers should implement disease management to minimize the risks posed by this pathogen.