First report of jujube anthracnose caused by Colletotrichum siamense in China.

Jujube (Zizyphus jujuba Mill.), a native small deciduous tree of China, is widely cultivated in China, Korea, India, Japan, Europe, and the United States (Chen et al. 2020). The fruit have been commonly consumed as healthy food supplements and traditional Chinese medicine for over 2000 years (Li et al. 2007). In August 2019, anthracnose-like leaf spot symptoms were observed on jujube plants in Xiaomenya Village, Jinan City, Shandong Province, China (36°27'39″N, 117°3'13″E), with over 30% leaf disease incidence. The spots were circular, sunken, brown in the center and with dark brown edges. As the spots enlarged and coalesced, it resulted in leaf perforation and early defoliation. Sometimes acervuli were observed on the lesions (Fig. S1a, b). To identify the causal agent, 20 diseased leaves were sampled, the margins of the lesions were cut into pieces (5 × 5 mm), sterilized and cultured following the protocol described previously (Wan et al. 2020) at 25 ℃ for 5 days. Twelve monospore isolates showing identical colony morphology were obtained. Three representative isolates, JNZG11, JNZG311, JNZG313, were used for further study. When grown on PDA the colony color was initially white and then turned pale-gray to gray in 5-day-old cultures. On the reverse, colonies were brown-black with an orange pigmentation near the center. Aerial mycelium was cottony, dense, white to pale-gray. Conidia were hyaline, 1-celled, smooth-walled, subcylindrical, oblong, attenuated with slightly rounded ends, (11.1-) 12.7-13.3 (-17.8) ×(-4.4) 5.2-5.5 (-6.3) µm (n=50). Appressoria were dark-brown, oval or irregular, (7.3-) 8.6-9.2 (-9.8) ×(-5.1) 5.8-6.9 (-7.0) µm (n=50) (Fig. S1c-g). The morphology resembled those of Colletotrichum gloeosporioides species complex (Cannon et al. 2012). For accurate identification, the sequences of the ribosomal internal transcribed spacer (ITS), actin (ACT), ß-tub2 (TUB2), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) of the 3 isolates were sequenced (Weir et al. 2012), and deposited into GenBank (Accession Nos. see Table 1). The six loci (ITS, GAPDH, ACT, CHS-1, CAL, and TUB2) were concatenated and the aligned sequences (1904 bp) were 99.7% homologous to ex-type C. siamense ICMP18578. The sequences of 38 Colletotrichum species (44 isolates) were downloaded from GenBank for phylogenetic analyses. In the maximum likelihood phylogenetic tree generated, the highest log likelihood was -8798.90 and the three isolates were all in the C. siamense clade (bootstrap support 94 %) (Fig. S2). To complete Koch's postulates, 60 healthy, mature jujube leaves on 12 branches (5 leaves per branch) (variety 'Zhongqiuhong') were inoculated with 20 µL of spore suspension (106 conidia/mL) or sterile water as a control. The branches were placed in sterile beakers containing a small amount of sterile water sealed with plastic wrap and maintained at 28 °C, 12 h light/dark. Five days after inoculation, all treated leaves showed the typical anthracnose symptom, similar to that observed in the field (Fig. S1h). The same fungus was re-isolated from the margins of the lesions using the aforementioned methods. Whereas no fungus were isolated from the controls. Previously, C. siamense has been reported to infect Z. mauritiana in China (Shu et al. 2020). To our knowledge, this is the first report of C. siamense causing anthracnose on Z. jujuba in China. This finding provides crucial information for the effective management of this disease.

First report of Alternaria alternata causing leaf spot on Hibiscus mutabilis in China.

Hibiscus mutabilis L. is a deciduous shrub native to China. Because of its ornamental value and ecological value, it has been widely cultivated in many provinces of China (Shang et al. 2020). In October 2021, leaf spot on Cotton rose with about 80% disease incidence was observed in Jinan (116.9408° N, 36.6688° E), Shandong, China. Symptoms first appear on leaves with small dark brown spots surrounded by yellow halos, then become irregular necrotic spots with yellow halos. The diseased leaf samples were packed in paper bags and transferred to the laboratory for isolation. The infected leaves were firstly surface-sterilized for 45 seconds in 75% ethanol, 1 min in 1% sodium hypochlorite, and 1 min in 75% ethanol, then rinsed for 2 min in distilled water and blotted on dry sterile filter paper. Then samples were cut into 5 × 5 mm pieces using a double-edge blade, and transferred onto the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L) and malt extract agar (MEA; 30 g malt extract, 5 g mycological peptone, 15 g agar per L), and incubated at 25 ◦C to obtain the pure culture. After 7 days of incubation, greyish fungal colonies appeared on PDA. Single-spore isolation method was employed to recover the pure cultures for six isolates. The colonies initially produce light gray aerial hyphae, which turn dark gray as they mature. Conidiophores (n=50) single or in small groups, straight or curved, sometime geniculate, 20-50 nm long, with scars. Conidia (n=50) were obclavate to pyriform and measured 15 to 60 µm long, 4 to 16 µm wide with 0 to 3 longitudinal, and 1 to 6 transverse septa with short beak (2-30 µm). The morphological characters matched those of Alternaria alternata (Simmons 2007). DNA was extracted from the fungal colonies using a Ezup Column Fungi Genomic DNA Purification Kit. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (tef1) were amplified with the primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999) and EF1-728F/EF1-986R (Carbone & Kohn 1999). The obtained sequences were deposited in the GenBank (ITS: OM759881 and OM759882, GAPDH: ON376732 and ON376733, tef1: ON376730 and ON376731). The morphological characteristics and molecular analyses of the isolate matched the descriptin of A. alternata. To perform pathogenicity test, The seedlings of twenty 2-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves and ten plants sprayed with sterile water served as control. The test was repeated three times. All plants were covered with polyethylene covers and kept under the greenhouse at 26 ± 1 ℃. After six days, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The fungal pathogen was reisolated from the artificially infected plants and confirmed as A. alternata based on morphocultural characteristics and PCR assays. The results indicated that A. alternata is a causal agent of the disease. The leaf spot disease of cotton rose caused by Nigrospora oryzae has already been reported from Sichuan, China (Han et al. 2021). To our knowledge, this is the first report on the presence of A. alternata affecting H. mutabilis plants. The identification could provide relevant information for adopting appropriate management strategies to control the disease.