Puccinia suaveolens Causing Leaf Rust on Cirsium setosum in China.

Thistle, Cirsium setosum (Willd.) M. Bieb., is widely distributed in China as a common weed in fields. It is also used as a traditional Chinese medicine for cooling blood, stopping bleeding, dispelling stasis, detoxifying, and resolving carbuncle. In 2023, we found a rust disease on plants of Cirsium setosum in the experimental field of Hebei Agricultural University, Baoding, Hebei Province, China, with incidence of 15% - 25% (Fig. S1 A, B). The diseased leaves turned yellow, and the leaf edges were slightly rolled. The yellow, oil-like pycnia and pycniospores covered the baxial surface of leaves, and brown pustules were produced after 2-3 weeks. On the adaxial surface of the leaves, the brown rust pustules were mainly along the leaf veins. Stems were also be infected later, and dark pustules were scattered. The diseased plants were relatively short and small, and produced relatively small or no flowers compared to healthy plants. A total of 100 plants with typical leaf rust symptoms and signs were collected. To confirm the pathogenicity, healthy plants of thistle were sprayed with 5 ml of urediospores suspension (2.6×105/ml), and plants sprayed with sterile distilled water were treated as control. The sprayed plants were incubated under high moist conditions at 18°C for 24 h, and the inoculated plants were grown at 20°C in a greenhouse. Ten days after inoculation, the plants inoculated with urediniospores showed rust symptoms with uredinia and urediniospores on the leaves (Fig. S1 C), while the control plants were healthy. For morphological characterization, urediospores were picked from the naturally infected plants and placed in a drop of sterile water on a glass slide using a sterile needle, and observed and measured under a microscope. Urediospores were nearly spherical, brown-yellow, and measured 15 - 25 µm in diameter (n=100) (Fig. S1 D). Telia were scattered on the baxial surface of the naturally infected leaves, and teliospores were oval, yellow-brown, double-celled, with very short hyaline pedicels, and measured 15-20 × 15-30 µm (n=100) (Fig. S1 E). For molecular characterization, about 200 µg of urediniospores was collected and placed in a 1.5 ml sterile centrifuge tube, and genomic DNA was extracted using the cetyl-trimethylammonium bromide method (Gawel et al. 1991). The internal transcribed spacer (ITS) region of the rDNA and the D1/D2 domain were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and NL1/NL4 (Borhani et al. 2013) in polymerase chain reaction (PCR), respectively. The PCR products were sequenced, and their sequences were aligned and compared with those deposited in GenBank. The obtained sequences were deposited in GenBank (OR600240 for ITS and OR598614 for D1/D2), which were 100% identical with 100% coverage to the ITS sequence (ON063373.1) and the D1/D2 sequence (ON063379.1) of Puccinia suaveolens (Menzies 1953). Based on the morphological characteristics and DNA sequences, the isolates were identified as P. suaveolens (Fig. S1 and Fig. S2). Thistle rust caused by Puccinia obtegens has been reported in some other parts of China (Zhang 2012). To the best of our knowledge, this is the first report of P. suaveolens causing leaf rust on C. setosum in China. This discovery is helpful for control of leaf rust on thistle grown for Chines medicine and other purposes, and the rust species could be used for biological control of thistle as a weed in crop fields.

First Report of Penicillium cellarum Causing Rot Disease on Dioscorea polystachya in China.

Dioscorea polystachya (Chinese yam) is a kind of medicine and food homologous crop, the tubers as its main production organ, with high potassium, low fiber, high protein and rich nutrition characteristics. In 2022, at the Chinese herbal medicine planting experimental site in Anguo, Baoding City, Hebei Province, China, we found the symptoms of Chinese yam decay during the storage, with an incidence of 15%~25%. The diseased part of Chinese yam tuber rots expands from the outside to the inside and sags, with a brown or dark brown discoloration, and the surface covered with a thick grayish green mold. The diseased tissue was first rinsed with clean water to remove dirts from the surface. Thereafter, 3 to 4 mm Chinese yam pieces were picked from rotting edge with a sterilized forceps, sterilized with 75% alcohol for 30 s followed by 0.1% mercuric chloride solution for 1min, and then rinsed three times with sterile water. The sterilized pieces were cultured on potato dextrose agar (PDA). One isolated fungus was obtained, and conidia were observed after incubation for 5 days at 26°C. Pure cultures were isolated by single-spore isolation. Conidia were single spore, round or oval, colorless. Conidiophores produce several rounds of symmetric or asymmetric small stems after multiple branches, which were shaped like brooms. The length and width of 100 conidia were measured, and size ranged from 3 to 4×3 to 4 µm. On the basis of morphological characteristics, the isolate was identified as Penicillium spp. (Uy et al. 2022). To further assess the identity of isolated species, the genomic DNA of the fungal isolate (SYRF1) was extracted by CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region and the ribosomal large subunit (LSU) were amplified and sequenced with primers ITS1/4, LR5/LROR respectively (White et al. 1990, Xu et al. 2010). The obtained ITS-rDNA region and LSU sequences (GenBank accession OQ707937 and OQ704185) of the isolate were more than 99% identity to the corresponding sequences of Penicillium cellarum in GenBank (KM249068 and MG714818). Phylogenetic results based on a maximum-likelihood analysis revealed that SYRF1 was grouped with P. cellarum. To determine the pathogenicity of the isolated fungi, tests were carried out by aseptic inoculation of fresh and healthy tubers. Before the experiment, the healthy tubers were washed, surface disinfected and dried. The tubers were then wounded with sterile inoculation needles, and the conidium-bearing hyphal discs (5 mm) were inoculated on the surface of the wounded tubers and covered with wet sterile cotton. Three tubers were inoculated repeatedly each time as the experimental group. Inoculate sterile PDA with three tubers as the control group. Each tuber was inoculated with four mycelium disks, and the pathogenicity test was repeated four times. The inoculated tubers were incubated at 26°C for 14 days with sterile PDA as control. After ten days, the inoculated points showed symptoms similar to those of the initial infection, whereas controls remained symptomless. The reisolated fungus matched SYRF1 based on morphological and sequence analyses, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of Penicillium cellarum as causative agent of postharvest rot of Chinese yam tubers in China. This finding will help inform the prevention and management of postharvest diseases of Chinese yam tubers.