First report of anthracnose of Lithocarpus polystachyus caused by Colletotrichum fructicola in China.

Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes because of the high content of flavonoids and dihydrochalcones in the leaves (Shang et al. 2022). In July 2022, the severe leaf lesion on L. polystachyus was first observed in Yongshun County, Xiangxi autonomous prefecture (28°45'34''N, 109°40'11''E), Hunan province, China. Yongshun County is characterized by hills and mountains, situated in a subtropical region with a mild and humid climate. A second outbreak in July 2023 was observed in the same area. The observed incident rates in the past two years were 87.3% and 90.6%, respectively. Once infected, almost all plant leaves will be infected, leading to a substantial reduction in the yield of L. polystachyus. The disease presented symptoms characterized by round or irregularly shaped lesions that initially manifested as brown spots. These lesions frequently merged into larger, dark-brown areas along the leaf margins before eventually wilting. To ascertain the pathogenic species responsible for this disease, fungal isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected with 75% ethanol and 0.1% HgCl then small pieces (1×1 cm), were placed onto potato dextrose agar (PDA) medium (Sigma-Aldrich, 70139) and incubated at 28°C for 6-9 days. Colonies were villiform and initially white, becoming gray after 6 days. Sterilized dissecting needles were used to pick single hyphal tips from the edge of the colonies and placed onto PDA for strain purification. After 15 days, the purified colonies grew fluffy white hyphae with abundant conidia. The conidia were cylindrical, had round ends, and ranged from 5.75 to 14.83 µm long and 1.75 to 2.38 µm wide (n=50). According to morphological and cultural characteristics, these isolates were preliminarily identified as Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Damm et al. 2012). To further affirm the identity of the pathogen, DNA was extracted from mycelia using a DNA extraction kit (Sigma-Aldrich, G2N70). The internal transcribed spacer (ITS) region, the transcription elongation factor (TEF), and the actin (ACT) gene were then amplified from genomic DNA extracted from three isolates (Cof1, Cof2, and Cof3) using specific primers. The primers utilized were ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and ACT-512F/ACT-783R (Carbone and Kohn 1999) for ITS region, transcription elongation factor gene and actin gene amplification, respectively. Sequence identity indicated that these isolates were highly homologous to C. fructicola. The ITS (Genbank No. PP002156, OR880553 and OR880554), TEF (No. PP061421, PP061422 and PP061423), and ACT (No. PP061418, PP061419 and PP061420) sequences of the isolates Cof1, Cof2, and Cof3 shared 99 to 100% identity with their counterparts (No. OR083309, MF627961, and OQ427895) in C. fructicola, respectively. A neighbor-joining phylogenetic tree constructed using MEGA11 (Tamura et al. 2021) also indicated that these isolates were C. fructicola. Both morphological and molecular characteristics confirmed the identification of this pathogen as C. fructicola. Colletotrichum species are known to cause anthracnose disease in a variety of economically important crops (Sharma and Kulshrestha 2015). To further validate the ability of the isolated C. fructicola to induce the same symptoms as observed in the field, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspensions (1×105 conidia per mL) from three isolates were individually inoculated onto artificially wounded leaves of 3-year-old L. polystachyus. Negative controls were established by inoculating leaf wounds with sterile distilled water. The plants were incubated in a greenhouse at 28°C and 90% humidity with a 12-h photoperiod. The experiment was replicated three times. Necrotic lesions were observed on all pathogen-inoculated wounds within 6 days after inoculation, whereas controls showed no observable symptoms. Morphological and molecular characterization of re-isolated pathogens from infected leaves indicated that the pathogens were identical. To our knowledge, this is the first report of anthracnose of L. polystachyus caused by C. fructicola in China. Farmers in the local mountainous areas are economically reliant on L. polystachyus production, while anthracnose has caused over half of the trees to lose their commercial value, resulting in significant economic losses. Our findings hold great promise for advancing strategies in the prevention and treatment of anthracnose in L. polystachyus.

First report of black patch disease caused by Phyllosticta capitalensis on Litsea cubeba in China.

Litsea cubeba, an economical important tree species originally from China, produces fruit from which essential oils are extracted and extensively used in the chemical industry (Zhang et al. 2020). In August 2021, a large-scale outbreak of black patch disease was first observed on the leaves of Litsea cubeba in Huaihua (27°33'N; 109°57'E), Hunan province, China (disease incidence 78%). A second outbreak in 2022, in the same area, lasted from June to August. Symptoms consisted of irregular lesions that initially appeared as small black patches near the lateral veins. These lesions grew along the lateral veins and formed feathery patches until almost the entire lateral veins of the leaves were infected by the pathogen. The infected plants grew poorly and eventually the leaves desiccated and the tree defoliated. To identify the causal agent, the pathogen was isolated from nine symptomatic leaves from three trees. Symptomatic leaves were washed with distilled water three times. Leaves were cut into small pieces (11 cm), surface sterilized with 75% ethanol for 10s and 0.1% HgCl2 for 3 min, and then washed 3 times in sterile distilled water. Surface disinfected leaf pieces were placed onto potato dextrose agar (PDA) medium with cephalothin (0.2 mg/ml) and incubated at 28°C for 4-8 days (about 16h light, 8h dark). Seven morphologically identical isolates were obtained, from which five were selected for further morphological examination and three for molecular identification and pathogenicity test. Strains from grayish white colonies with a granular surface and grayish black wavy edges; bottom of the colonies turned black over time. Conidia were hyaline and nearly elliptical, unicellular. The sizes of conidia ranged from 8.59 to 15.06 µm (n=50) in length and 3.57 to 6.36 µm (n=50) in width. These morphological characteristics are consistent with the description of Phyllosticta capitalensis (Guarnaccia et al. 2017, Wikee et al. 2013). To further confirm the identity of this pathogen, genomic DNA of three isolates (phy1, phy2 and phy3) were extracted to amplify the internal transcribed spacer (ITS) region, the 18S rDNA region, the transcription elongation factor (TEF), and actin (ACT) gene with ITS1/ITS4 (Cheng et al. 2019), NS1/NS8 (Zhan et al. 2014), EF1-728F/EF1-986R (Druzhinina et al. 2005) and ACT-512F/ACT-783R (Wikee et al. 2013) primers, respectively. Sequence similarity indicated that these isolates were highly homologous to Phyllosticta capitalensis. The ITS (Genbank No. OP863032, ON714650 and OP863033), 18S rDNA (Genbank No. OP863038, ON778575 and OP863039), TEF (Genbank No. OP905580, OP905581 and OP905582) and ACT (Genbank No. OP897308, OP897309 and OP897310) sequences of isolates Phy1, Phy2 and Phy3 shared up to 99%, 99%, 100% and 100% similarities with their counterparts (Genbank No. OP163688, MH051003, ON246258 and KY855652) in Phyllosticta capitalensis, respectively. To further confirm their identity, a neighbor-joining phylogenetic tree was generated using MEGA7. Based on morphological characteristics and sequence analysis, the three strains were identified as P. capitalensis. To fulfill Koch's postulates, conidial suspension (1×105 conidia per mL) collected from three isolates were independently inoculated on artificially wounded detached leaves and leaves on trees of Litsea cubeba. Leaves were inoculated with sterile distilled water as negative controls. The experiment was repeated three times. All pathogen-inoculated wounds exhibited necrotic lesions within 5 days on detached leaves and 10 days on the leaves growing on trees after inoculation, whereas no symptoms were observed on the controls. The pathogen was exclusively re-isolated from the infected leaves and showed identical morphological characteristics to those of the original pathogens. P. capitalensis is a destructive plant pathogen that has been shown to cause leaf spots or black patch symptoms on variety of host plants around the world (Wikee et al. 2013), including oil palm (Elaeis guineensis Jacq.), tea plant (Camellia sinensis), Rubus chingii and castor (Ricinus communis L.). To our knowledge, this is the first report of black patch disease of Litsea cubeba caused by P. capitalensis in China. This disease causes severe leaf abscission in fruit development stage of Litsea cubeba and leads to a large amount of fruit drop.

First report of leaf spot disease caused by Fusarium oxysporum on Litsea cubeba in China.

Litsea cubeba, an important industrial plant species that originated in China, produces fruit essential oil extensively applied in the chemical industry (Xiang et al. 2020). In July 2020, a large-scale outbreak of leaf spot disease on Litsea cubeba was first observed and then monitored over time in Yueyang (29°37'N; 113°13'E) and Changsha (28°06'N; 113°02'E), Hunan province, China. Symptoms of this disease consisted of round-shaped lesions that initially appeared as small light-brown spots. With the increase in number, these small spots coalesced into larger, dark-brown lesions leading to yellowing and abscission of the leaves. To identify the causal agent this disease, the pathogen was isolated with a tissue separation method (Gao et al. 2020). The infected leaf tissues surface-disinfected with 75% ethanol and 0.1% HgCl were aseptically cut into small pieces (11 cm) and then placed onto potato dextrose agar (PDA) medium with cephalothin (0.2 mg/ml) and incubated at 28°C for 3-5 days. The purified colonies on PDA exhibited fluffy white hyphae, secreted a dark red pigment that had been observed in previous studies (Xiao et al. 2015) and produced microconidia and macroconidia. The microconidia were single-celled, non-septate, ovoid, and ranged from 3.08 to 13.89 µm long and 2.17 to 3.62 µm wide (n=50). Macroconidia were three to five-septate, slightly curved, and ranged from 11.77 to 26.85 µm long and 3.31 to 4.50 µm wide (n=50). These morphological features suggested that theisolates were most likely Fusarium oxysporum (Savian et al. 2021). To further confirm the identity of this pathogen (designated as Fox-1), the TEF-1a gene (Genbank accession No. OM281065) and rDNA ITS region (Genbank accession No. OM250084) were cloned and then sequenced (Cui et al, 2021). Sequence alignments indicated that the ITS and TEF-1a sequences shared 99.8% (504/505) and 99.7% (665/667) similarities with that of F. oxysporum (Genbank accession No. MF667966, KT230848), respectively. Both of the morphological characteristics and molecular data were used to identify this pathogen as F. oxysporum Schltdl.: Fr. 1824. To further verify whether these isolates of F. oxysporum can cause leaf spot disease, Koch's postulates were tested (Gradmann 2014). The purified pathogens were inoculated on artificial wounds of detached Litsea cubeba leaves and the leaves on the field plants of Litsea cubeba, respectively. The wounds of leaves were inoculated with sterile distilled water as negative controls. The experiment was performed independently three times, each with three leaves and three inoculated wounds on each leaf. All pathogen-inoculated wounds developed dark brown or black lesions on detached leaves within 3 days and on leaves on plants within 9 days, whereas the controls showed no symptoms. Re-isolations from infected leaves confirmed that the re-isolated pathogens possessed identical morphological characteristics to those of the original pathogens. To our knowledge, this is the first report of leaf spot infection of Litsea cubeba caused by F. oxysporum in China. This disease severely delays plant development and significantly decreases the yield of essential oil of Litsea cubeba. Our results laid a foundation for the subsequent research into pathogenic mechanisms drug sensitivity tests, which will contribute to the prevention and cure of leaf spot disease of Litsea cubeba. References: Cui, L. X., et al. 2021. Plant Dis. 105:7. Gao, W., et al. 2020. Plant Dis. 105:501. Gradmann. 2014. J. Microbes Infect. 16:885-892. Savian, L. G., et al. 2021. Plant Dis. 104:1870. Xiang, Y. J., et al. 2020. J. Chin. Cereals Oils Assco. 35:186-195. Xiao, J. L., et al. 2015. Hunan Agric. Sci. 4:105-108.