First Report of Black Spot Disease Caused by <i>Diaporthe compacta</i> on <i>Cynanchum rostellatum</i> in China

The plant species of Cynanchum rostellatum (Apocynaceae; syn: Metaplexis japonica) is widely distributed in China. Because of its multiple active medicinal components in roots, fruit husks and seeds, and therapeutic properties, it is a common traditional Chinese herb (Li et al., 2022). In September 2023, black punctate lesions were observed on the leaves and fruit husks of the plant in roadside shrubs at Dalian Minzu University (39°2'50"N, 121°46'27"E), Liaoning Province, China. Additionally, extensive black necrotic patches grew on leaves, fruits and stems. Initial infections appeared as small black spots on the host's epidermal tissues. As the disease progressed, the adjacent lesions coalesced, forming larger blackened areas. In severe cases, the infected stems became entirely blackened, with branching nodes exhibiting high susceptibility. Microscopic examination revealed that the pathogen primarily colonized superficial host cells without invading vascular bundles or internal fruit husk tissues. On PDA, the fungal isolates initially produced white aerial hyphae after 3–4 days of incubation. After one week the hyphae proliferated, forming a dense mycelial mat. The colony reverse initially appeared yellow, but gradually became brownish-black. After approximately 40 days, mature black perithecia，with aggregated beak-like ostioles developed within the colony. Conidiomata observed on host tissues were globose to ovoid, unilocular, and embedded in stromata, measuring 95-350 µm in diam.. Conidiogenous cells were phialidic, cylindrical, with an enlarged base and a slightly tapered apex, measuring 6.5–12.3 × 1.3–2.5 µm. Two types of conidia were observed, α-conidia were aseptate, fusiform, hyaline, 5.5‒8.0 × 1.8‒3.0 µm and biguttulate; β-conidia were filiform, curved, smooth, 21.5–25 × 1.0–1.5 mm. Conidiogenous cells subcylindrical, phialidic, terminal, slightly tapering towards the apex, 6.5‒12.5 × 1.3‒2.8 mm. These morphological features align with the descriptions of the anamophic Phomopsis charateristics of Diaporthe compacta (Gao et al., 2016). In order to molecularly identify the fungal isolate LM-0035, a multiple locus DNA sequences of Internal Transcribed Spacer (ITS), Elongation Factor 1-alpha (EF-1α), β-tubulin (TUB), histone H3 (HIS), and calmodulin (CAL) were amplified for the fungus by using the primer pairs of ITS1/ITS4(White et al. 1990), EF-F/TEF-R, TI/BT2b(Carbone et al.,1999), CYLH3F/H3-1b(Glass, 1995), and CAL-228F/CAL-737R(Carbone et al.,1999), respectively. The resulting sequences were deposited in GenBank under the accession numbers PV864833 (ITS), PV768881 (EF-1α), PV768880 (TUB), PV768879 (HIS), and PV768878 (CAL). The phylogenetic analysis revealed that the isolated LM-0035 clustered together with several reference strains of D. compacta (e.g., SKJ1, LC3076, ZJUE0403) with strong bootstrap support (100%), indicating a close evolutionary relationship with D. compacta. The phylogenetic tree also clearly delineated other reference isolates into distinct clades corresponding to D. sojae, D. unshiuensis, D. biguttulata, D. sennae, and D. eres, each supported by high bootstrap values. These results demonstrate that the topology of the phylogenetic tree is robust and the phylogenetic relationships are reliable. The outgroup strain Diaporthella corylina effectively rooted the tree and supported the overall structure of the phylogeny. The phylogenetic analysis revealed that isolate LM-0035 is D. compacta. For pathogenicity assessment, the fungal isolate LM-0035 was inoculated onto healthy stems and leaves of Cynanchum rostellatum in vitro (with three replicates and one control). Chlorosis and yellowing of the leaves were observed obviously after 4 days of inoculation, followed by browning and wilting of the entire leaf after 7 days. two weeks later, black granular structures (conidiomata) began to form along the main vascular bundles on both sides. Inoculation on stems showed visible infection after 3 days, with typical near-elliptical brown lesions and surrounded by water-soaked halos after two weeks. These lesions rapidly extended along the stem, reaching about 1.5 cm in length. The re-isolating procedure confirmed the original pathogenic fungus. This fungus was also reported on Camellia in China (Gao et al., 2016). To our knowledge, this is the first report of D. compacta on Cynanchum rostellatum in China. The identification of this fungal pathogen provides a theoretical guidance for the disease management.