First Report of Penicillium subrubescens Causing Root Rot of Knoxia roxburghii in China.

Knoxia roxburghii (syn. Knoxia valerianoides), locally known as 'Zi Daji', is a perennial herb that belongs to the Rubiaceae family, cultivated in different areas of China and recognized for its medicinal properties in traditional Chinese medicine (Chen et al. 2022). In 2021, root rot was observed during summer on K. roxburghii in Xiangyun and Dali, Yunnan Province (25°25'N, 100°40'E), China. Root rots were characterized by dark brown tissue from stem base to root, loss of vitality in tender leaves and wither. Three symptomatic root samples of K. roxburghii collected from different fields were rinsed with running water, and 0.5-1 cm2 fragments of diseased tissues were cut and surface-disinfested with 75% ethanol for 30 s, and 1% NaClO for 180 s. The fragments were then washed with sterile water, transferred to potato dextrose agar (PDA, 4.6%) and incubated at 28℃ in the dark for 3 days. A total of 13 isolates with consistent appearances were obtained by single spore isolation. These colonies on PDA showed gray and light brown obverse, and light green reverse after 10 days. The average growth rate was 3 mm per day. Conidia were nearly spherical or broadly elliptic, greyish-green, and 1.4-2.4×1.3-2.2 µm in size (n=50). The conidiophore has symmetrical or asymmetrical broom branches from the tip, with 2-4 small stems. The conidiophore branching patterns were predominantly biverticillate; stipes coarsely roughened, 80-210×2.6-3.0 µm; metulae were usually appressed verticils of 3-6, 6.4-12.5×1.6-3.0 µm; phialides were short and wide neck, 3.8-4.8×1.1-1.8 µm (n=30). The morphological characteristics of the fungus were identical to Penicillium (Mansouri et al. 2013). To further identify the isolate, one isolate (ByF10) was randomly selected for identification. DNA was extracted from mycelia using a simplified CTAB method. Primer pairs, ITS1/ITS4 and Bt2a/Bt2b were used to amplify the partial regions of rDNA internal transcribed spacer (ITS) and ß-tubulin (TUB), respectively (White et al. 1990; Glass and Donaldson 1999). Blast searches showed that the sequences of ITS (OQ954757) and TUB (OQ970059) of isolate ByF10 were 99% (MH865456) and 100% (KC797611) identical with P. subrubescens CBS 130205 and CBS 129617, respectively. A concatenated phylogenetic tree (ITS+TUB) constructed using the maximum likelihood method showed that ByF10 was closely grouped next to isolates of P. subrubescens. Pathogenicity test was carried out using 1-year-old healthy seedlings of K. roxburghii cv. Yunji-1 growing on autoclaved soil (n=10). Ten plants were inoculated with mycelial blocks (5 mm2), which were taken from the colony margins of a 10-day-old culture (PDA) colony, and placed on the roots near the soil. Five control plants were inoculated with non-colonized PDA plugs. The pathogenicity test was repeated three times. All plants were kept at 25℃, 70% relative air humidity, and 12 h light/12 h regime dark for 35 days. After that period 95% of inoculated plants showed typical symptoms of root browning. P. subrubescens was only re-isolated from the inoculated plants, and identified based on morphological and molecular characteristics. No symptoms were observed in the controls. To the best of our knowledge, this is the first report of P. subrubescens causing root rot on K. roxburghii in China and the world.