Occurrence of leaf spot disease on Arundo donax caused by Pyricularia oryzae (synonym: Magnaporthe oryzae ) in China

Arundo donax is a tall perennial herbaceous species within the Poaceae family. It serves roles in ecological restoration, as industrial raw materials and biomass energy (Ortega et al. 2024). In March 2025, leaf spot symptoms were observed in an A. donax nursery, located in Shishou city (30°35’N, 112°13’E), Hubei province, China. Disease incidence on leaves was 20 to 30%. The leaf lesions were typically spindle-shaped, or elliptical, gray to light brown in the center with clear dark water-soaked like margins, sometimes with shallow yellowing halos. Later, the spots gradually expanded and coalesced causing leaf blight. Symptomatic leaf samples were collected, surface-sterilized (75% ethanol for 30 s, followed by 1% NaOCl for 2 min, and rinsed three times with sterile distilled water), cut into small pieces containing both diseased and healthy margins, and plated onto potato dextrose agar (PDA). The plates were incubated at 25°C in darkness for 3–5 days. Two single-spore isolates (YzU 251265 and YzU 251266) were obtained following the method of Jia (2009) were used for subsequent morphological and molecular identification. The colonies on PDA were circular, white to smoke grey in the center, greyish sepia at the edge. To stimulate the sporulation, strains were grown on oatmeal agar (OA) at 25°C for 7-10 days under a 12 h near-ultaviolet light/12 h dark cycle. After 7 to 10 days, the upcoming conidia were pyriform, hyaline, comprising two septa, and range in size from 20.08 to 34.49 × 6.97 to 10.33 µm. On the basis of morphology, it was identified as Pyricularia sp. (Klaubauf et al. 2014). The species was further confirmed via sequence analysis. Genomic DNA was extracted from fresh mycelia using CTAB method described in Watanabe et al. (2010). The internal transcribed spacer of (ITS) of ribosomal DNA, calmodulin (CAL), and actin (ACT) gene regions were PCR amplified using ITS5/ITS4 (White et al. 1990), CAL-228F/CAL-737R, and ACT-512F/ACT-783R (Carbone and Kohn 1999) primer pairs, respectively. BLASTn analysis of the ITS, CAL, and ACT gene sequences from strain YzU 251265 (GenBank accession nos. PX736389, PX754579, and PX754578, respectively) revealed 100% identity with the corresponding sequences of Pyricularia oryzae (ITS:LC876990; CAL: MT767933; ACT: CP099701). A phylogenetic tree was constructed based on combined ITS- ACT - CAL sequences using maximum likelihood (ML) analysis in MEGA v.7.0.26. The analysis revealed that our isolates clustered with P. oryzae reference strains with 100% bootstrap support. Based on the morphological and molecular characteristics, the isolated fungus was identified as P. oryzae. The pathogenicity was evaluated by both in vitro and in vivo assays. A spore suspension (10⁵ spores/mL) was amended with 0.02% Tween-20 solution. In vitro, spore suspension was applied to needle-wounded site of Arundo donax leaves (10 µL, n=3/leave) and spray to whole living plants (n=3), which were all maintained with high humidity and room temperature. Control plants were treated identically with sterile water. After inoculation, plants were covered with pre-moistened black bags and kept overnight in the greenhouse, then transferred to a growth chamber (25°C; 12 h light/12 h dark) for cultivation. The assay was performed in three independent replicates. Seven days post-inoculation, plants in the in vitro assay developed lesions similar to field symptoms, while those in the in vivo assay showed small necrotic spots. All control plants remained asymptomatic. The re-isolation was verified via morphology for fulfilling Koch's postulates. P. oryzae can induce symptoms up to 50 gramineous species, including rice, wheat, barley, oats, wild millet, foxtail millet, and others (Chiapello et al. 2012). To our knowledge, this is the first report of leaf spot disease on A. donax caused by P. oryzae worldwide, which will help for developing effective strategies of disease management.