First Report of Leaf Spot of Strobilanthes cusia Caused by Aspergillus niger in Guangdong, China

Abstract

Strobilanthes cusia is a significant economic plant in China, valued for its medicinal properties and as a crucial raw material for industrial extraction of indigo pigment. In September 2025, a leaf spot disease was observed on mature, cultivated S. cusia plants in a farm located in Maoming City, Guangdong Province, China (21°41'11" N, 110°29'12" E), where the plants were grown in monoculture with sprinkler irrigation, received regular fertilization, and no pesticides were applied. Approximately 15% of plants were infected in the field. Symptoms initially presented as irregular brown necrotic lesions accompanied by chlorotic halos, often resulting in leaf necrosis and abscission. Symptomatic leaf tissues from 9 diseased leaves were cut into 5 mm × 5 mm segments from the margins of lesions, surface-disinfested in 75% ethanol for 30 s and 1% NaClO for 2 min, rinsed 3 times with sterile distilled water, and plated onto potato dextrose agar (PDA). Plates were incubated at 28°C in the dark. The pathogen was consistently isolated from 9 leaves, yielding 8 morphologically similar isolates via hyphal-tip purification. On PDA, the 8 isolates all formed brownish-black, granular colonies. Conidiophores were smooth and hyaline. Conidial heads were radiate with spherical vesicles (82.59 ± 12.46 µm, n=30). Conidia were brown to black, spherical to subglobose (3.96 ± 0.42 µm, n=30). Based on morphology, they were preliminarily identified as Aspergillus sp. (Raper and Fennell 1965). The isolates named Aspergillus sp. A1~8 were subjected to DNA sequencing of ITS (the internal transcribed spacer), cmdA (calmodulin), and BenA (β-tubulin). Concatenated sequence analysis and morphological characteristics confirmed that all 8 isolates belonged to the same species, and a representative isolate was designated TK1 (A6). All 24 sequences in e-Xtra were submitted to GenBank. To more accurately resolve the taxonomic placement of TK1, a separate phylogenetic tree was reconstructed for it. ITS, BenA, and cmdA genes were amplified using primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and cmd5/cmd6 (Houbraken et al. 2007), respectively. The obtained sequences were deposited in GenBank under accession numbers PX671824 (ITS), PX712781 (BenA), and PX677349 (cmdA). BLASTn analysis of the calmodulin (cmdA) sequence revealed high identity with reference strains of Aspergillus niger, specifically 534/535 bp (>99% identity) with LC813239, MK193878, LC830490, PX562648, and MK193877. In the phylogenetic tree based on concatenated sequences, the isolate clustered with reference strains of A. niger with 98% bootstrap support. Pathogenicity was tested on healthy S. cusia plants (n=3). 5 leaves per plant were selected. For the experimental group, the leaves were inoculated with a conidial suspension (1 × 10⁶ spores/mL), while for the control group (n=3), the leaves were inoculated with sterile water. All plants were maintained in a growth chamber at 32°C, 90% RH, with a 12-h photoperiod. After 9 days, typical leaf spot symptoms, identical to those in the field, developed exclusively on inoculated leaves. Controls remained symptomless. A. niger was not recovered from controls, but was reisolated from symptomatic tissue and morphologically confirmed as identical to the original TK1 isolate, fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf spot of Strobilanthes cusia caused by Aspergillus niger.