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Leaf spot and dieback were observed on box elder (Acer negundo) grown in a nursery in Tai’an city, Shandong Province, China, in 2019, with a disease incidence of 86%. The incidences of Exserohilum rostratum isolation were 75% from the shoots and 66.6% from the leaves of field-infected plants. Isolates were identified at the species level on the basis of morphological characteristics and through phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) region and cam, gapdh, tef1, rpb2, tub2, and his genes from the Exserohilum isolates. The effects of temperature on the mycelial growth of the Exserohilum rostratum isolates were also characterized. In greenhouse tests, seedlings inoculated with the pathogen exhibited systemic symptoms similar to those observed in the field. In pathogenicity experiments on shoots, wounded seedlings were observed to be blighted, suggesting that leaf spot and dieback may develop into more severe blight or dieback when high winds, sudden temperature decreases, or insect infestations occur. To our knowledge, this is the first report of dieback and leaf spot caused by E. rostratum on a species of A. negundo.

Cyclocarya paliurus, an important endangered plant in China, has considerable medicinal, timber, and horticultural value. However, little is known about diseases that affect its health. In recent years, stem canker diseases on C. paliurus have been observed frequently in newly established nurseries in Jiangsu Province, China. Symptomatic trees showed elliptical, sunken lesions on the bark, with internal discoloration, leading to enlarging cankers with delineated margins. Pathogenicity tests with fungi isolated from symptomatic samples reproduced typical canker symptoms on both detached branches and potted plants of C. paliurus. Moreover, conidia from pycnidia of isolate ZB-23 could also cause stem canker on C. paliurus. Through combined morphological observation and DNA sequences of ITS region, β-tubulin, and translation elongation factor 1-α genes, the pathogen was identified as Botryosphaeria dothidea. Multigene maximum likelihood and maximum parsimony phylogenetic analyses further supported the identification of the pathogen. To our knowledge, this is the first report of B. dothidea causing stem canker on C. paliurus in China.

Cyclocarya paliurus is an extremely valuable and multifunctional tree species whose leaves have traditionally been used in used in medicine or as a medicinal tea in China. In recent years, anthracnose has been frequently observed on young leaves of C. paliurus in several nurseries located in Jiangsu Province, resulting in great yield and quality losses. To date, no information is available about the prevalence of C. paliurus anthracnose in China. The main purpose of the present study was to characterize the etiology of C. paliurus anthracnose. Phylogenetic analysis of the eight-loci concatenated dataset revealed that all 44 single-spore Colletotrichum isolates belonged to three species in the Colletotrichum gloeosporioides species complex, namely, Colletotrichum aenigma, Colletotrichum fructicola, and C. gloeosporioides sensu stricto. Phenotypic features, including the colony appearance and the morphology of conidia, appressoria, and ascospores, were consistent with the phylogenetic grouping. Virulence tests validated that the three Colletotrichum species could cause typical symptoms of anthracnose on C. paliurus leaves, similar to those observed in the field. The optimum mycelial growth temperature ranged from 25 to 30°C for all representative isolates, while C. gloeosporioides s. s. isolates exhibited greater tolerance to high temperature (40°C). Fungicide sensitivity assays indicated that all three Colletotrichum species were sensitive to tetramycin, which may be a potential alternative for the management of C. paliurus anthracnose. To our knowledge, this study provides the first report of C. aenigma, C. fructicola, and C. gloeosporioides s. s. causing C. paliurus anthracnose in China as well as in the world.

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch's postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

American sweetgum (Liquidambar styraciflua L.) is an important tree for landscaping and wood processing. In recent years, leaf spots on American sweetgum with disease incidence of about 53% were observed in about 1200 full grown plants in a field (about 8 ha) located in Pizhou, Jiangsu Province, China. Initially, dense reddish-brown spots appeared on both old and new leaves. Later, the spots expanded into dark brown lesions with yellow halos. Symptomatic leaf samples from different trees were collected and processed in the laboratory. For pathogen isolation, leaf sections (4×4mm) removed from the lesion margin were surface sterilized with 75% ethanol for 20s and then sterilized in 2% NaOCl for 30s, rinsed three times in sterile distilled water, incubated on potato dextrose agar (PDA) at 25 °C in the darkness. After 5 days of cultivation, the pure culture was obtained by single spore separation. 6 isolate samples from different leaves named FXA1 to FXA6 shared nearly identical morphological features. The isolate FXA1 (codes CFCC 54675) was deposited in the China Center for Type Culture Collection. On the PDA, the colonies were light yellow with dense mycelium, rough margin, and reverse brownish yellow. Conidiophores (23-35 × 6-10 µm) (n=60) were solitary, straight to flexuous. Conidia (19-34 × 10-21 µm) (n=60) were single, muriform, oblong, mid to deep brown, with 1 to 6 transverse septa. These morphological characteristics resemble Stemphylium eturmiunum (Simmons 2001). Genomic DNA was extracted from mycelium following the CTAB method. The ITS region, gapdh, and cmdA genes were amplified and sequenced with the primers ITS5/ITS4 (Woudenberg et al. 2017), gpd1/gpd2 (Berbee et al. 1999), and CALDF1/CALDR2 (Lawrence et al. 2013), respectively. A maximum likelihood phylogenetic analysis based on ITS, gapdh and cmdA (accession nos. MT898502-MT898507, MT902342-MT902347, MT902336-MT902341) sequences using MEGA 7.0 revealed that the isolates were placed in the same clade as S. eturmiunum with 98% bootstrap support. All seedlings for pathogenicity tests were enclosed in plastic transparent incubators to maintain high relative humidity (90%-100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. For pathogenicity, the conidial suspension (105 spores/ml) of each isolate was sprayed respectively onto healthy leaves of L. styraciflua potted seedlings (2-year-old, 3 replicate plants per isolate). As a control, 3 seedlings were sprayed with sterile distilled water. After 7 days, dense reddish-brown spots were observed on all inoculated leaves. In another set of tests, healthy plants (3 leaves per plant, 3 replicate plants per isolate) were wound-inoculated with mycelial plugs (4×4mm) and inoculated with sterile PDA plugs as a control. After 7 days, brown lesions with light yellow halo were observed on all inoculation sites with the mycelial plugs. Controls remained asymptomatic in the entire experiment. The pathogen was reisolated from symptomatic tissues and identified as S. eturmiunum but was not recovered from the control. The experiment was repeated twice with the similar results, fulfilling Koch's postulates. S. eturmiunum had been reported on tomato (Andersen et al. 2004), wheat (Poursafar et al. 2016), garlic (L. Fu et al. 2019) but not on woody plant leaves. To our knowledge, this is the first report of S. eturmiunum causing leaf spot on L. styraciflua in the world. This disease poses a potential threat to American sweetgum and wheat in Pizhou.

Cultivated as a medicinal plant, Cyclocarya paliurus is a monotypic species in its genus and native to China. However, limited information is available regarding diseases that affect its health. In recent years, severe foliar blight and occasional dieback of C. paliurus were observed in the provinces of Anhui, Guangxi, Hubei and Jiangsu in China. The fungus Nigrospora sphaerica was consistently isolated from 70 diseased samples, and its identity was confirmed based on phylogenetic and morphological studies. The phylogenetic analyses of the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF), and beta-tubulin (TUB) genes compared 15 representative isolates with reference specimens within the Nigrospora genus and validated the identity of the pathogenic fungus. The pathogenicity tests of C. paliurus leaves showed that regardless of whether mycelial plugs or conidial suspensions were used, inoculation with the N. sphaerica isolates resulted in foliar blight symptoms similar to those observed under field conditions. Mycelial growth of N. sphaerica occurred at temperatures ranging from 5 to 35 °C, with an optimum between 25 and 30 °C. In addition, high relative humidity (>85%) could promote conidial germination of N. sphaerica. This study represents an initial step in studying N. sphaerica as a new phytopathogen of C. paliurus in China with the goal of developing efficient control strategies.