First Report of <i>Aspergillus flavus</i> Causing Root Rot on Peony (<i>Paeonia suffruticosa</i>) in Tongling, China

Peony (Paeonia suffruticosa Andr) is an ornamental and medicinal plant in China. The root cortex of peony has been used in traditional Chinese medicine for alleviating fever and dissipating blood stasis. The medicinal peony is mainly produced in Tongling, Anhui Province, China, and it is also called "Fengdan". In November 2021, we collected peony plants with root rot disease from several fields in Tongling county, Anhui Province, China (118°0'51" E, 30°48'11" N). Approximately 20 to 40 percent of peony plants were affected in the fields, which caused a decline in the quality and yield of this plant. The plant sample with rotted and blacked root was rinsed in tap water to remove soil debris, and small pieces (5 mm×5 mm) were cut from the diseased tissues, disinfected with 0.5% NaClO solution and 75% ethanol for 5 min, rinsed in sterilized distilled water three times, and placed on potato dextrose agar (PDA) medium at 28°C. Ten isolates with different morphological characteristics were obtained from the diseased tissues. Among the isolates, Pleiocarpon algeriense B4 was confirmed to be a pathogenic agent inducing peony root rot in our previous work (Ni et al. 2023). And another pure isolate B9 colony was yellow-green, loose in structure and soft to cotton-like in texture. Microscopic examination showed that conidia were spherical with smooth or rough surface, and diameter of 2.34 - 6.85 μm (n = 20). Based on these morphological characteristics, the isolates are consistent with the description of the genus Aspergillus (Samson et al. 2014). To confirm the identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and the calmodulin (CaM) genes were amplified using the primer pair ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Hong et al. 2006), and CMD5/CMD6 (Glass et al. 1995), respectively. The sequence lengths obtained by amplification were 551, 566 and 613 bp, respectively. Sequences were deposited in GenBenk (OP810685, ITS; OQ753104, TUB2; OQ753105, CaM). BLAST analysis showed the ITS, TUB2, CaM sequences of B9 were 100.00%, 100.00% and 99.14% homology with those of A. flavus NB16 (MF685311, ITS); A. flavus CMXY30197 (MG991395, TUB2) and A. flavus CMXY8113 (MG991523, CaM), respectively. A phylogenetic tree was constructed using MEGA11 software based on ITS, TUB2, and CaM sequences. The results indicated that B9 strain was closely clustered with the reference strain of A. flavus. Thus, based on morphological and molecular characteristics, B9 was identified as A. flavus. Pathogenicity tests were performed on one-year-old peony seedlings. The ten healthy peonies were inoculated with 50 mL of suspension (1 × 108 CFU/mL). Ten healthy peonies irrigated with 50 mL of sterile water were used as control pots. After one-month, root rot symptoms appeared on inoculated plants, whereas control plants remained asymptomatic. To perform Koch's postulates, the fungus was reisolated from the diseased roots and identified as A. flavus. To our knowledge, this is the first report of peony root rot caused by A. flavus in Tongling, China. This species was previously reported causing fruit rot on kiwifruit in China (Zhu et al. 2022) and grapes in Pakistan (Zhu et al. 2020). This finding should be considered for developing efficient management strategies for peony root rot disease.