First Report of Root Rot Caused by <i>Plectosphaerella cucumerina</i> on <i>Indigofera pseudotinctoria</i> in China

Indigofera pseudotinctoria is an economically important perennial shrub in the family Fabaceae, mainly distributed in southwestern China. This plant has been used in traditional medicine for its natural flavonoids, polyphenols, and amino acids and also serves as a highly nutritious forage legume for livestock, containing 21.82% crude protein, 2.7% crude fat, and 10.54% crude fiber (Gerometta et al. 2020; Xu et al. 2025). In June 2024, root rot disease was observed on I. pseudotinctoria in commercial plantings in Renyi Town, Rongchang District, Chongqing, China. In infected plants, the aboveground parts exhibited yellowing and necrosis, while the tap root showed varying degrees of decay with black-brown lesions. The disease incidence in the three surveyed fields ranged from 15% to 30%. Symptomatic root tissues were excised from the six plants and were surface sterilized in 75% ethanol for 1 min, followed by 1% NaClO for 2 min, and rinsed three times with sterile water, then dried on sterilized paper, plated on potato dextrose agar (PDA) plates, and incubated at 25°C in the dark. After 5 days, hyphal tips of the growing colonies were transferred onto PDA plates for purification. Totally, thirty-six isolates were obtained belonging to nine fungal species. Eight of the isolates resembled the genus Plectosphaerella. Colonies of these isolates were white, moist, with appressed mycelium. The hyphae were transparent and branched. The conidia were hyaline, smooth, thin-walled, mostly elliptical to ovoid or cylindrical, 0–1 septate, and occasionally curved, and were 3.7-9.4 × 1.9-4.4 μm in size (n=50). The internal transcribed spacer (ITS) region, translation elongation facter 1 alpha (EF1-α), and large subunit rRNA (LSU) gene of three representative isolates were amplified and sequenced using ITS1/ITS4, EF1-983F/EF-2218R, LR0R/LR7 (Giraldo and Crous 2019; Hua et al. 2025), respectively. The sequences of the three isolates were 100% identical, and sequences of one representative isolate MJPC3 were deposited in GenBank (PX488016, ITS; PX531816, EF-1α; and PX488017, LSU). BLASTn homology searches for these sequences revealed 99 to 100% identity to the ITS (LR026797, 490/490 bp), the EF-1α (LR026492, 783/787 bp), and the LSU (LR025935, 827/827 bp) of P. cucumerina CBS 137.33 from Nicotiana tabacum in England (Giraldo and Crous 2019). The combined phylogenetic tree (ITS+EF1-α+LSU) of Plectosphaerella species constructed using IQ-TREE showed that the representative isolates belonged to the P. cucumerina clade. Based on the morphological characteristics and molecular analysis, the fungus was identified as Plectosphaerella cucumerina (Giraldo and Crous 2019). To test the pathogenicity, six-week-old I. pseudotinctoria seedlings were planted individually in pots containing sterilized soil and inoculated with 40 mL of conidial suspension (10⁶ conidia/mL) of isolate MJPC3, while seedlings treated with sterilized water served as controls. Eight inoculated plants and eight controls were maintained in a greenhouse at 25°C and 80% relative humidity. The pathogenicity test was repeated twice. One week after inoculation, similar symptoms were observed on inoculated plants, whereas control plants remained healthy. Plectosphaerella cucumerina was 100% reisolated from symptomatic tissue. Plectosphaerella cucumerina has been reported to cause root rot on various host plants worldwide, including alfalfa, common bean, fennel, and lettuce (Cai et al. 2021; Mihajlović et al. 2024; Yang et al. 2018; Zhao et al. 2021). This is the first report of P. cucumerina causing root rot on I. pseudotinctoria in China, providing a foundation for further disease control.