First report of Phaeosphaeria caricicola causing leaf spot on Belamcanda chinensis in China.

Belamcanda chinensis (L.) Redouté, a member of the Iridaceae family, is globally well-known for its medicinal value as clearing away heat, detoxifying, detumescence and pain (Qin 2000). In 2021, spots were observed on 40% B. chinensis leaves and about 28 disease index in Wanzhou District (30°32'N; 108°22'E) of Chongqing. Initial symptoms appeared as circular yellow white, sunken spots lesions, and then expanded into irregular lesions, the center of the spots was beige, external layer was light brown and surrounded by yellow halo. Symptomatic leaf tissues (5 × 5 mm) were cut from the infected margin, surface sterilized with 75% ethanol for 1 min, washed with 3% sodium hypochlorite for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium incubated at 25°C for 7 days in the dark, forty isolates with similar morphology were obtained. Three isolates (SG9、SG20 and SG33) was selected for subsequent research. Colonies color changed from beige to light brown color after 14 days on PDA medium. Fungal colonies transformed from beige to brown at the edges after 28 days and light brown on top. Ascomata dark brown, ellipsoidal to globose 116.6 to 253.3 × 89.6 to 172.6 µm in diamensions. Asci stipitate, cylindrical with obtuse ends, and 69.1 to 114.7 × 10.2 to 24.1 µm (n = 30) in size, with eight overlapping linearly biseriate ascospores. Ascospores brown, narrowly fusiform, straight or slightly curved with three transversely septate, slightly constricted at septa, and 9.7 to 12.6 × 27.6 to 32.6 µm (n = 30). These characteristics are consistent with Phaeosphaeria sp. reported by Quaedvlieg et al in 2013. DNA was extracted from representative isolates. The internal transcribed spacer (ITS) region, the large subunit rDNA (LSU), the small subunit rDNA (SSU) and the RNA polymerase II second largest subunit (RPB2) genes were amplified for Polymerase chain reaction (PCR) by used ITS1/ITS4, LR5/LROR, NS1/NS4, and RPB2-5f2/RPB2-7cr primers (White et al. 1990; Vilgalys et al. 1990; Qi M W. et al. 2008; De G. J. et al. 1992). The sequences were submitted to NCBI GenBank: SG-G9 (ITS, OR701701; LSU, OR701699; SSU, OR701700; RPB2, OR738464); SG-G20 (ITS, OQ748032; LSU, OQ780728; SSU, OQ780723; RPB2, OQ779979); SG-G33 (ITS, OQ748033; LSU, OQ780729; SSU, OQ780722; RPB2, OQ779980). A phylogenetic analysis revealed a 99% similarity to the Phaeosphaeria caricicola CBS 603.86 (ITS, KF251182; LSU, GQ387590; SSU, GQ387529; RPB2, KF252189) sequences. Mycelial agar plugs (5-mm diameter) from a 7-day-old PDA culture of a fungal isolate were placed onto pinpricked leaves of three two-year-old B. chinensis plants. While the sterile PDA plugs inoculated in pinpricked leaves of B. chinensis as controls. Inoculated plants were placed in a greenhouse at 25°C and remained 95±1% relative humidity. The inoculated leaves of treatment developed symptoms after 20 days, whereas no symptoms occurred on controls, fulfilling Koch's postulates. The experiments were repeated three times. The fungus was re-isolated and was identical to original isolate by morphologically and molecularly. As far as we know, P. caricicola can cause diseases on carex plants and has been found in Switzerland. This is the first report of P. caricicola causing leaf spot on B. chinensis in China. Along with recording the occurrence of this disease, plant disease management strategies need to be established to reduce losses.

First Report of Fusarium ipomoeae Causing leaf rot on Hosta plantaginea in China.

Hosta plantaginea is an important horticultural plant with ornamental value and is widely cultivated in China. Since April 2022, leaf rot has been observed in the H. plantaginea plants in Wanzhou District, Chongqing City, China (31º14'58"N, 108º53'25"E), the initial symptom is a yellow and brown lesion on the edge of the leaf, in the late stage, brown blighted tissue caused leaves to curl and abscise. Ten typical diseased leaves were collected, the margins of infected tissues were cut into small pieces (5×5 mm) and were sterilized in 75% Ethanol for 30 s, 3% sodium hypochlorite for 3 min, rinsed three times with sterile water, then dried on sterile filter paper and placed to potato dextrose agar (PDA) medium at 25℃for 4 days. Thirteen isolates with morphological characteristics similar to those of Fusarium spp. (Nelson et al. 1983) were recovered. These isolates had white, pink and yellowish mycelia, two isolates produced irregular colonies, and remaining isolates showed round. Two of each type were selected for intensive study (yz2, yz11, yz9 and yz17). The colony of yz2 reached 62 mm in diameter on PDA medium after seven days, macroconidia were elongated sickle-shaped, 3-5 septa, and 12.92 to 21.49 × 3.42 to 5.90 µm in size, microconidia were oval and measured 5.69 to 12.95 × 3.41 to 9.80 µm in size, conidiophores were whorled and branched, yz9 attained 74 mm in diameter after nine days, macroconidia were curved sickle-shaped and apex cell acuminate, 26.9 to 57.2 × 2.4 to 7.1 µm, 3-5 septa. The microconidia were fusiform, 17.8 to 28.8 × 11.2 to 14.5 µm. Conidiophores variable in length. Genomic DNA was extracted from 7-day-old aerial mycelia of four strains (yz2, yz9, yz11 and yz17). The internal transcribed spacer (ITS) region (White et al. 1990), translation elongation factor (EF-1α) (Cao et al. 2014) and partial RNA polymerase second largest subunit (RPB2) (Wang et al. 2019) gene regions were amplified and multilocus phylogenetic analysis was conducted, their sequences were deposited in NCBI Genbank with the following accession numbers: the strains of yz2 and yz11 with OQ829372 and OR236201 for ITS, OQ848594 and OR282462 for EF-1α, OR492296 and OR492297 for RPB2; yz9 and yz17 with OQ829383 and OR236222 for ITS, OQ848595 and OR282463 for EF-1α, OR492295 and OR492298 for RPB2. The ModelFinder was used to select the best-fit model in PhyloSuite v1.2.2, the Bayesian Inference method (BI) analysis was used to estimate the system relationship, yz9 and yz17 were identified as Fusarium ipomoeae, yz2 and yz11 were identified as Fusarium tricinctum. To verify Koch's postulates, 8 healthy plants of H. plantaginea (two-year-old) grown were rinsed with sterile water, after 5 leaves per plant were stabbed with a sterilized needle, 4 plants were inoculated with conidial suspension (1×106 conidia mL-1), other plants injected with sterile water as control, then placed in a greenhouse maintained with 95% relative humidity at 25 ± 1°C. The symptoms on the leaves were similar to field after inoculation for 7 days, whereas all control leaves remained healthy. The same pathogen was re-isolated and re-identified based on multilocus phylogenetic analysis, fulfilling Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing leaf rot on H. plantaginea in China. In addition, F. ipomoeae was reported to cause leaf spot in Peanut (Xu et al. 2021), and F. tricinctum can cause fruit rot on navel orange in China (Yang et al. 2023). H. plantaginea as a horticultural plant is popular with some people, but it has long been threatened by Fusarium.spp. The finding can provide a theoretical basis for control leaf rot on H. plantaginea.