First Report of <i>Macrophomina phaseolina</i> Causing Root Rot on <i>Pogostemon cablin</i> in China

Patchouli (Pogostemon cablin (Blanco) Benth.) is an important medicinal plant belonging to the family Lamiaceae, which is native to South and Southeast Asia. It is renowned for its detoxification, antibacterial and other pharmacological properties (Pan et al. 2024), and is widely used in the flavor and fragrance industry. In July 2023, severe symptoms of root rot were observed on P. cablin in a farmland of Liantang Town in Zhaoqing, Guangdong Province, China (23°1’32.52’’N, 112°27’28.8’’E). Symptoms initially appeared as black irregular plaques on the root epidermis, and gradually spread to the inside of the root, and then decayed. The surface of the stem base also turned black until the roots were unable to transport water or nutrients to the aboveground parts. The plants eventually died with branches and leaves withered (Fig.1). The disease incidence was 90% in three fields of 3.3 acres, and 9 infected plants were randomly collected for pathogen isolation, with 3 plants sampled per field. Symptomatic roots were cut with sterile scissors into small pieces, surface-disinfected by 1% NaClO (1 min) and 75% ethanol (1 min), washed three times with sterile distilled water, then cultured on potato dextrose agar (PDA) with kanamycin (100 μg/ml) and ampicillin (100 μg/ml), and incubated at 28°C in the dark for 5 days. After purification using hyphal-tip method (Leslie and Summerell. 2006), two fungal isolates G2 and G8 were obtained, and G8 was selected for further study. The colony of G8 grown on PDA was white at the beginning of growth, and then gradually turned grayish-white, finally appeared black (Fig.2). Microsclerotia that formed after 5 days were black, round to ovate, and measured 43.12 to 215.12 μm in length×38.03 to 148.02 μm in width (n=30) (Fig.3). To observe conidiophore and conidia, strain G8 was cultured on oat meal agar (OMA) with UV irradiation for 30 minutes (Zhao et al. 2020). After incubation at 28°C in the dark for 10 to 15 days, conidia were observed to be unicellular, hyaline, round or oblong in shape. These morphological features were consistent with the description of Macrophomina phaseolina (Huda-Shakirah et al. 2019). The internal transcribed spacer (ITS) regions, 28S large subunit ribosomal RNA (LSU) region and actin gene (ACT) were amplified and sequenced using primer sets ITS1/ITS4, NL1/NL4 (White et al. 1990) and ACT-512F/ACT-783R (Carbone and Kohn 1999). Sequences were deposited in GenBank (ITS: PV688484.1 and PV688510.1; LSU: PX850623.1; and ACT: PX921628) and analyzed on NCBI for BLASTn alignment, which showed 99.81% to 100% identity with M. phaseolina (ITS: PV605624.1; LSU: MH877909.1; and ACT: KF951820.1) (Fig.5). Pathogenicity testing was performed three times on 5-month-old healthy P. cablin grown in plastic pot with soil. Barley seeds (150 g) were soaked in distilled water overnight and autoclaved, then inoculated with two discs (6 mm in diameter) of G8, which had grown on PDA for 5 days. After incubation at 28°C in the dark for two weeks, the seeds were dried at 37°C for 7 days and then ground (El_Komy et al. 2020). Barley seeds inoculum (80 g) was placed around each plant at the root, and the typical root rot symptoms were observed after 7 days, while the control group did not show any symptoms (Fig.4). The fungus that was reisolated from symptomatic P. cablin roots was morphologically and molecularly identical to M. phaseolina, while no pathogens were isolated from the control group, thus fulfilling Koch’s postulates. M. phaseolina has been reported to cause leaf blight on Pometia pinnata in Malaysia (Khoo et al. 2023), and this is the first report of M. phaseolina causing root rot on P. cablin in China and worldwide.