First report of Alternaria alternata causing leaf blight on Bidens pilosa in China

Bidens pilosa L., commonly known as Spanish needle, is an invasive weed in China. Its invasiveness and environmental adaptability are strong, resulting in yield losses to grain and threatening a billion dollar pork industry in China that relies on that grain (Shang and Zhu 2019). In April 2025, plants of B. pilosa showing leaf spots were observed in Zhanjiang, China. The disease incidence was above 80% of plants investigated in the field (1 ha). Symptoms on leaves primarily appeared as small brown spots (3 to 5 mm diam) with yellow halos. As the symptoms progressed, necrotic lesions expanded to produce large, dark brown spots with irregular shape. Ten symptomatic leaves from 10 plants were collected. Small pieces of tissue (4 × 4 mm) were cut from lesion borders and were surfaced disinfected in 75% ethanol for 30 s, followed by 1 min in 1% NaClO, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C with a 12-h photoperiod. After 5 days, ten morphologically similar fungal isolates were obtained from all 10 leaf samples and three representative strains (GZC-1, GZC-2 and GZC-3) were used for morphological and molecular characterization. Colonies were brown with cottony aerial mycelium. Conidia were solitary or in chains, ovoid to obclavate, 3-7 transverse and 0-3 longitudinal septa, with darker in septa, usually 22.19-48.07×5.17-10.27 μm in size, with a beak length of 0 to 7.25 μm (n=50). These morphological characteristics were consistent with the description of Alternaria alternata (Simmons 2007). The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1α), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and RNA polymerase II second largest subunit (rpb2) were amplified and submitted to GenBank with primers ITS1/ITS4，EF1-728F/EF1-986R，GDF1/GDR1 and fRPB2-5F/fRPB2-5R (Carbone and Kohn 1999). The ITS (PX057423-25), tef1α (PX068176-78), gapdh (PX068180-82) and rpb2 (PX068185-87) sequences were 100% identical to the A. alternata type strain CBS 916.96 (accession nos. AF347031, KC584634, AY278808 and JQ811951, respectively). A phylogenetic tree was generated using the concatenated sequences of ITS, tef1α, gapdh and rpb2. All three isolates are clustered with A. alternata type strain CBS 916.96. For pathogenicity tests, all of the three isolates were inoculated to B. pilosa plants (n=10) by spraying conidial suspension (105 conidia/ml) on the surface of leaves. Mock-inoculated controls (n=10) were sprayed with sterile distilled water. All plants were wrapped in polyethylene bags for 24 h and incubated at 28°C in a growth chamber at 90% relative humidity. After 7 days, all inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic. A. alternata was re-isolated from the lesions and the identity of the fungus was confirmed based on morphology and molecular characterization. The pathogenicity test was repeated three times with similar results. A. alternata has been reported to cause diseases in a broad range of hosts (Sanahuja et al. 2018; Taycir et al. 2023), but not in B. pilosa. This is the first report of A. alternata causing leaf blight on B. pilosa in China. A. alternata can be used as a potential biological control agent to limit the expansion of invasive weed B. pilosa and save the grain yield in China. Additional research could identify or clarify the mycoherbicide value of the pathogen to offset the risk to the pork industry by decreasing feed supply.