Mendelian randomization analysis reveals the impact of physical and occupational activities on the risk of gastroesophageal reflux disease and Barrett's esophagus.

BACKGROUND: Recent studies have indicated that participating in physical activity may provide a safeguard against gastroesophageal reflux disease (GERD). Nevertheless, the precise links between physical and occupational activity and the occurrence of GERD and Barrett's esophagus (BE) are still uncertain. METHODS: Conducting univariate and multivariate Mendelian randomization investigations to examine the causal relationship between exposures and outcomes. Genetic variation simulation was used in randomized experiments. Data on physical and occupational activity were obtained from the UK Biobank and GWAS catalog. In the meantime, data on GERD and BE were extracted from a high quality meta-analysis. RESULTS: The results of univariate Mendelian randomization analysis using multiple methods suggest a causal relationship between strenuous sports or other forms of exercise (as a protective factor) and GERD/BE. At the same time, three types of occupational related physical activities, including heavy manual or physical work, shift work and walking or standing work, are risk factors for GERD/BE and have a causal relationship with them. These results were reconfirmed through multivariate Mendelian randomization analysis, which excluding the influence of other potential confounding factors. CONCLUSIONS: The findings indicated that strenuous sports or other forms of exercise could lower the likelihood of GERD/BE, while excessive physical strain in the workplace, prolonged periods of standing or walking, and shift work could raise the risk of GERD/BE. Acknowledging this risk and implementing suitable measures can contribute to the prevention of GERD and BE, thus mitigating the associated health burden.

Colletotrichum nanjingense sp. nov. and C. gloeosporioides s.s. Causing Leaf Tip Blight on Jasminum mesnyi in Nanjing, Jiangsu, China.

Jasminum mesnyi Hance is an important medicinal and ornamental plant. This species is native to South Central China and Vietnam and grows primarily in the subtropical biomes. In June 2022, 17 Colletotrichum strains were isolated from leaf tip blight on foliage of J. mesnyi in Nanjing, Jiangsu, China. Based on morphological characteristics and multilocus phylogenetic analyses of six genomic loci (ITS, CAL, ACT, TUB2, CHS-1, and GAPDH), a new species, namely, C. nanjingense, and a known species, namely, C. gloeosporioides s.s., were described and reported. Pathogenicity tests revealed that both species were pathogens causing leaf tip blight on J. mesnyi. The results provided necessary information for disease control and enhanced our understanding of the diversity of Colletotrichum species in China.

Identification and Fungicide Screening of Fungal Species Associated with Walnut Anthracnose in Shaanxi and Liaoning Provinces, China.

Walnut is cultivated around the world for its precious woody nut and edible oil. Recently, walnut infected by Colletotrichum spp. resulted in a great yield and quality loss. In August and September 2014, walnut fruits with anthracnose were sampled from two commercial orchards in Shaanxi and Liaoning provinces, and five representative isolates were used in this study. To identify the pathogen properly, four genes per region (internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, actin, and chitin synthase) were sequenced and used in phylogenetic studies. Based on multilocus phylogenetic analysis, five isolates clustered with Colletotrichum fioriniae, including its ex-type, with 100% bootstrap support. The results of multilocus phylogenetic analyses, morphology, and pathogenicity confirmed that C. fioriniae was one of the walnut anthracnose pathogens in China. All 13 fungicides tested inhibited mycelial growth and spore germination. Flusilazole, fluazinam, prochloraz, and pyraclostrobin showed the strongest suppressive effects on the mycelial growth than the others, the average EC50 values ranged from 0.09 to 0.40 µg/ml, and there was not any significant difference (P < 0.05). Pyraclostrobin, thiram, and azoxystrobin were the most effective fungicides on spore germination (P < 0.05), and the EC50 values ranged from 0.01 to 0.44 µg/ml. Pyraclostrobin, azoxystrobin, fluazinam, flusilazole, mancozeb, thiram, and prochloraz exhibited a good control effect on walnut anthracnose caused by C. fioriniae, and preventive activities were greater than curative activities. Pyraclostrobin at 250 a.i. µg/ml and fluazinam at 500 a.i. µg/ml provided the highest preventive and curative efficacy, and the values ranged from 81.3 to 82.2% and from 72.9 to 73.6%, respectively. As a consequence, mancozeb and thiram could be used at the preinfection stage, and pyraclostrobin, azoxystrobin, flusilazole, fluazinam, and prochloraz could be used at the early stage for effective prevention and control of walnut anthracnose caused by C. fioriniae. The results will provide more significant instructions for controlling the disease effectively in northern China.

First report of Diaporthe acuta causing leaf blight of Acer palmatum in China.

Acer palmatum Thunb. is an important ornamental deciduous tree with colorful foliage, and widely cultivated in Japan, Korea and China (Carlos et al. 2016). In October 2021, a foliar disease of ~95% incidence was observed on A. palmatum in three community parks, Shaoxing, Xuzhou, and Wuhan cities, China. The symptoms appeared as brown necrotic lesions at the tips, margin, and surface of leaves. Thirty leaves with symptoms from three trees were collected from the three parks. Small pieces (3 to 5 mm2) cut from the lesion margins were placed on potato dextrose agar (PDA) after surface-sterilized and incubated at 25°C in the dark, following the protocol described previously (Wan et al. 2022). The same fungus was isolated from 31% of 150 tissue pieces. Pure cultures were obtained from the tip of hyphae. Three representative isolates (WH52, SX13, and XZ96) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelia, cottony, and the reverse was white. Gray pycnidia developed on the sterile alfalfa stems at 25°C with a 14/10 h light/dark cycle in 30 days. Conidiophores were hyaline, cylindrical, septate, branched, smooth, 14.3-37.2 × 1.5-3.7 µm (n = 30). Conidiogenous cells were cylindrical, 5.6-21.6 × 1.3-2.1 µm (n = 30). Alpha conidia were aseptate, fusiform to oval, 6.5 ± 0.6 × 2.2 ± 0.2 µm (n = 50), bi- or multi-guttulate. Beta conidia were aseptate, hyaline, and curved, 31.0 ± 3.5 × 1.0 ± 0.1 µm (n = 30). Gamma conidia were aseptate, infrequent, botuliform, 12.4 ± 1.2 × 1.4 ± 0.1 µm (n = 10). Morphological characteristics of the three isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of the three isolates was extracted and the internal transcribed spacer region (ITS), histone H3 (HIS), partial translation elongation factor 1-alpha (TEF1-α), beta-tubulin (TUB), and calmodulin (CAL) genes were amplified with primers ITS1/ITS4 (White et al. 1990), CYLH3F/H3-1b (Glass and Donaldson, 1995; Crous et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), Bt2a/Bt2b (Glass and Donaldson 1995), and CAL-228F/CAL-737R (Carbone et al. 1999), respectively. The genomic DNA sequences were deposited in GenBank with Accession Nos. OP522005, OP522447, OP522448, and OP566419 to OP566430 (Supplementary Table 1). BLAST search of the sequences from the three isolates showed high similarities with sequences of Diaporthe acuta Y.S. Guo & G.P. Wang (ex-type PSCG 047). BLAST results were listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed WH52, SX13, and XZ96 in the clade of D. acuta. Based on the phylogeny and morphology, the three isolates were identified as D. acuta. The pathogenicity was tested on potted 3-yr-old seedlings of A. palmatum. Healthy leaves wounded with a sterile needle (1 mm in diameter) were inoculated with 5-mm plugs from the edge of 3-day-old culture of the three isolates. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags to maintain humidity in a greenhouse at the day/night temperatures at 25 ± 2°C. The plastic bags were removed on the fifth day. Five days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe acuta was reisolated from the lesions on the inoculated leaves and was confirmed based on morphological characteristics and ITS sequence analyses. No fungus was isolated from the controls. Diaporthe acuta was previously reported to cause pear shoot canker in China (Guo, et al. 2020), and D. foliicola, D. monospora and D. nanjingensis caused leaf blight of A. palmatum (Wan et al. 2022). This is the first report of D. acuta causing leaf blight of A. palmatum. This finding will provide an effective basis for developing control strategies for the disease.

Unravelling Species Diversity and Pathogenicity of Colletotrichum Associated with Anthracnose on Osmanthus fragrans in Quanjiao, China.

Osmanthus fragrans is a popular ornamental tree species known for its fragrant flowers and is widely cultivated in Asia, Europe, and North America. Anthracnose is a disastrous threat to the growth and development of O. fragrans and has caused significant economic losses. To reveal the potential pathogen diversity of anthracnose, 127 isolates of Colletotrichum were isolated from the symptomatic leaves. Morphological studies and multilocus phylogenetic analyses with the concatenated sequences of the internal transcribed spacer, glyceraldehyde-3-phosphate dehydrogenase, chitin synthase, actin, beta-tubulin, calmodulin, and the intergenic region between Apn2 and Mat1-2-1, as well as a pairwise homoplasy index, test placed the causal fungi as two new species, Colletotrichum anhuiense (two isolates) and C. osmanthicola (12 isolates), and three known taxa, C. fructicola (18 isolates), C. gloeosporioides (62 isolates), and C. karstii (33 isolates). Among them, C. gloeosporioides was the most dominant, and C. anhuiense was occasionally discovered from the host tissues. Pathogenicity tests in vivo on O. fragrans leaves revealed a significant difference in virulence among these species. Of them, C. gloeosporioides, C. osmanthicola, and C. anhuiense were significantly more virulent than C. fructicola and C. karstii, while C. karstii was the least virulent. To our knowledge, this study was the first to report the pathogen diversity of anthracnose on O. fragrans.

Three New Species of Diaporthe Causing Leaf Blight on Acer palmatum in China.

Diaporthe spp. are often reported as plant pathogens, endophytes, and saprobes. In this study, three new species (Diaporthe foliicola, D. monospora, and D. nanjingensis) on Acer palmatum were described and illustrated based on morphological characteristics and phylogenetic analyses. Phylogenetic relationships of the new species were determined by multilocus phylogenetic analyses based on partial sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF), ß-tubulin (TUB), histone H3 (HIS), and calmodulin (CAL) genes. Genealogical concordance phylogenetic species recognition with a pairwise homoplasy index test was used to verify the conclusions of the phylogenetic analyses. All species were illustrated and their morphology and phylogenetic relationships with other related Diaporthe spp. are discussed. In addition, the tests of Koch's postulates showed that the three new species were pathogens causing leaf blight on A. palmatum.

Protein Phosphatase CgPpz1 Regulates Potassium Uptake, Stress Responses, and Plant Infection in Colletotrichum gloeosporioides.

Protein phosphatases play important roles in the regulation of various cellular processes in eukaryotes. The ascomycete Colletotrichum gloeosporioides is a causal agent of anthracnose disease on some important crops and trees. In this study, CgPPZ1, a protein phosphate gene and a homolog of yeast PPZ1, was identified in C. gloeosporioides. Targeted gene deletion showed that CgPpz1 was important for vegetative growth and asexual development, conidial germination, and plant infection. Cytological examinations revealed that CgPpz1 was localized to the cytoplasm. The ΔCgppz1 mutant was hypersensitive to osmotic stresses, cell wall stressors, and oxidative stressors. Taken together, our results indicated that CgPpz1 plays an important role in the fungal development and virulence of C. gloeosporioides and the multiple stress responses generated.

Diaporthe sapindicola sp. nov. Causes Leaf Spots of Sapindus mukorossi in China.

Sapindus mukorossi Gaertn. (Sapindaceae), or soapberry, is an important biodiesel tree in southern China. In recent years, leaf spot disease on soapberry has been observed frequently in a soapberry germplasm repository in Jianning County, Sanming City, Fujian province, China. The symptoms initially appeared as irregular, small, yellow spots, and the centers of the lesions became dark brown with time. Three fungal isolates from lesions were collected. Koch's postulates were performed, and their pathogenicity was confirmed. Morphologically, α-conidia from diseased tissues were single-celled, hyaline, smooth, clavate or ellipsoidal, and biguttulate, measuring 6.2 to 7.2 × 2.3 to 2.7 µm. In addition, the three isolates in this study developed three types (α, ß, and γ) of conidia on potato dextrose agar, and their morphological characteristics matched those of Diaporthe. A phylogenetic analysis based on internal transcribed spacer, TEF, TUB, HIS, and CAL sequence data determined that the three isolates are a new species of Diaporthe. Based on both morphological and phylogenetic analyses, the causal fungus, Diaporthe sapindicola sp. nov., was described and illustrated.

Pathogenicity and Biological Characteristics of Septotinia populiperda Causing Leaf Blotch of Willow.

Salix babylonica is an important landscape tree in China and has been widely planted. In this study, the pathogenicity of Septotinia populiperda causing leaf blotch of Sa. babylonica to four willow species (Sa. matsudana, Sa. chaeomoloides, Sa. matsudana f. tortuosa, and Sa. suchowensis) and Populus tomentosa (Chinese white poplar) was determined. Its sexual stage and biological characteristics were studied. Leaves from four willow species and P. tomentosa were inoculated with mycelial plugs. Typical leaf blotches with sporodochia were produced on all inoculated leaves. Among the isolates studied, some developed conidia but sclerotia were rare. The sclerotia developed apothecia after induction at 4°C for 3 months in an incubator and 2 more months outdoors from January to March. The biological characteristics of S. populiperda showed that mycelium grew better on complete medium than on potato dextrose agar, Czapek's agar, and minimal medium. For mycelial growth, the optimal carbon source was dextrose and the optimal nitrogen source was yeast powder. Conidia germination rate was 59.4% at 24 h. The conidia germinated best in a 4% willow leaf extraction. The optimal temperature for conidia germination was 25°C, and the optimal pH was 4.

First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 µm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 µm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 µm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 µm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 µm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 µm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 µm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), ß-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, ßt2a/ßt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.

First Report of Diaporthe eres Causing Leaf Spot of Viburnum odoratissimum var. awabuki in China.

Viburnum odoratissimum var. awabuki (K. Koch) Zabel ex Rumpl. is an evergreen tree, used as a landscape plant in China. In June 2019, a foliar disease of ~60% incidence was observed on V. odoratissimum var. awabuki at the campus of Nanjing Forestry University, Jiangsu, China. The symptoms were initially irregular small red-brown spots, later enlarged and became brown to black. Small pieces of tissue (3 to 4 mm2) cut from lesion margins were surfaced sterilized in 75% ethanol for 30 s and 1.5% NaClO for 60 s, then rinsed in sterile water and placed on potato dextrose agar (PDA) at 25℃. Pure cultures were obtained from the tip of hyphae. Using the standard phytopathological procedure, two representative isolates (SH161 and SH181) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelium, radiate, and the reverse was white. Black pycnidia developed on the sterilized alfalfa stems at 25°C with a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, straight to sinuous, 9.4 to 26.0 × 1.0 to 2.5 µm (n=30). Conidiogenous cells were 2.1 to 15.1 × 0.9 to 2.5 µm (n=30). Alpha conidia were 7.4 ± 0.6 × 2.0 ± 0.2 µm (n=50), hyaline, ellipsoidal to lanceolate. Beta conidia were 29.5 ± 1.8 × 1.1 ± 0.1 µm (n=30), aseptate, hyaline, smooth, curved to hooked. Morphological features of two isolates matched those of Diaporthe spp.. DNA of two isolates was extracted and the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, ßt2a/ßt2b and CYLH3F/H3-1b. The sequences were deposited into GenBank (Accession Nos. for isolate SH161: OK326730 for ITS, OK413403 to OK413406 for TUB, CAL, HIS and TEF1-α; and isolate SH181: OK331347 for ITS, OK413407 to OK413410 for TUB, CAL, HIS, and TEF1-α). BLAST search of SH161 showed high similarities with sequences of Diaporthe eres (AR5193) [KJ210529 (ITS), Identities = 438/512, (94%); KJ420850 (HIS), Identities = 466/472, (99%); KJ210550 (TEF1-α), Identities = 345/350, (99%); KJ434999 (CAL), Identities = 344/345, (99%); KJ420799 (TUB), Identities = 508/517, (98%)]. BLAST results of SH181 are listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed SH161 and SH181 in the clade of D. eres. Based on the multi-locus phylogeny and morphology, two isolates were identified as D. eres. The pathogenicity was tested on 1-yr-old cuttings of V. odoratissimum var. awabuki in the greenhouse. Healthy leaves were wounded with a sterile needle, then inoculated with 5-mm plugs from the edge of two isolates cultures. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags bidaily to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Three days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe eres was reisolated from inoculated leaves. No fungus was isolated from controls. Diaporthe eres was reported from Viburnum lantana in Austria. Also, it was reported from V. odoratissimum and V. tinus in Ukraine. This is the first report of D. eres causing V. odoratissimum var. awabuki leaf spots in China. This finding will provide an effective basis for developing control strategies for the disease.

First Report of Erysiphe alphitoides Causing Powdery Mildew of Cocculus orbiculatus in China.

Cocculus orbiculatus (L.) DC. (Menispermaceae) is a vine traditionally used as a medicinal herb in Asia and grows primarily in wet tropical biomes (POWO 2022). In late April 2022, typical symptoms of powdery mildew were observed on leaves of C. orbiculatus on the campus of Nanjing Forestry University, China. Approximately 90% of the plants were infected. Superficial mycelia and conidia were amphigenous on the leaves, pale yellow, and severe infections caused necrotic discoloration of the leaves. Infected leaves were collected to identify the pathogen. Hyphae were hyaline and branched. Conidiophores were solidary, unbranched, straight, cylindrical, smooth, hyaline, 69.3 ± 11.1 × 7.9 ± 0.6 µm, (n = 50). Foot cells were mostly cylindrical, straight, rarely curved, smooth, hyaline, 53.2 ± 6.2 × 7.5 ± 0.4 µm, (n = 50). Appressoria were lobulate, solitary or in opposite pairs, hyaline to pale yellow. Conidia were single, ellipsoid, oval or doliform, hyaline or pale yellow, 38.6 ± 2.3 × 20.9 ± 0.8 µm, (n = 50). Conidial germ tubes developed at a subterminal position. No chasmothecia were observed. Representative specimens were deposited in the NJFU Herbarium (NF50000010). Based on these morphological characteristics, this fungus (MFJ 1-1) was provisionally identified as Erysiphe alphitoides (Takamatsu et al. 2007). To verify the identification of the pathogen, mycelia and conidia were obtained from diseased leaves and genomic DNA of the fungus (MFJ 1-1) was extracted. The internal transcribed spacer region (ITS) and large subunit (LSU) gene were amplified with primers ITS1/ITS4 and LR0R/LR5, respectively (White et al. 1990, Rehner and Samuels 1994). The sequences were deposited in GenBank (ON612134 for ITS, ON620080 for LSU). BLAST results showed that the ITS and LSU sequences were highly similar to E. alphitoides [ITS: KF734882, identities = 632/633 (99%) LSU: MK357414, identities = 890/893 (99%)]. Phylogenetic analyses with the concatenated sequences using Bayesian inference and maximum likelihood placed the isolate in the clade of Erysiphe alphitoides. Pathogenicity was confirmed by gently pressing the infected leaves onto five leaves per plant, and three healthy plants were inoculated. Three uninoculated plants served as controls. The plants were placed in a growth chamber with a 16 h photoperiod at 22 ± 2°C, 70% of relative humidity. Symptoms developed 10 days after inoculation, whereas the control leaves remained symptomless. The powdery mildew developing on the inoculated plants was identified to be E. alphitoides based on morphological characters and ITS sequences. This fungus has a worldwide distribution and a broad host range. Recently, Ipomoea obscura (Pan et al. 2020) and Aegle marmelos (Banerjee et al. 2020) have been found to be additional hosts. To our knowledge, this is the first report of powdery mildew caused by E. alphitoides on C. orbiculatus in the world. This finding provides crucial information for developing effective strategies to monitor and manage this disease.

Leaf Spots of Salix babylonica Caused by Colletotrichum gloeosporioides s.s. and C. siamense Newly Reported in China.

Salix babylonica L. shows a great potential for restoration of contaminated water or soils and has a high ornamental value (Li et al. 2015). In mid-October 2021, a leaf spot disease, with an incidence of approximately 61%, occurred on leaves of 25-year-old S. babylonica on the campus of Nanjing Forestry University. On average, 65% of the leaves per tree were infected. Symptoms began as dark brown, irregular spots, and the centers were grayish white. The spots gradually enlarged with time. Fresh specimens were collected from 3 trees (10 leaves/tree). Small tissue pieces cut from lesion margins were surface-sterilized (Mao et al. 2021), plated on potato dextrose agar (PDA), and incubated at 25°C. Three representative isolates (NL1-7, NL1-10, and NL1-13) were obtained and deposited in The China Forestry Culture Collection Center. The colonies of 3 isolates were white, grayish white at the center. The conidia of 3 isolates were one-celled, straight, subcylindrical, hyaline, smooth, 14.6-18.6 × 4.3-6.7 µm, 13.8-16.7 × 4.7-6.0 µm and 12.1-16.9 × 5.4-7.5 µm (n = 50) for NL1-7, NL1-10, and NL1-13, respectively. The conidiophores of NL1-7 were hyaline to pale brown, septate, and branched, 18.9-48.0 µm (n = 50). Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled. The conidiophores and appressoria of the other two isolates were almost identical to NL1-7. Based on morphological characteristics, the 3 isolates matched the Colletotrichum gloeosporioides species complex (Weir et al. 2012). DNA of the 3 isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin 2 (TUB2) loci were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-354R, GDF1/GDR1, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. ON870951 and ON858477 to ON858481 for NL1-7; ON908707 and ON858482 to ON858486 for NL1-10; ON870949 and ON858487 to ON858491 for NL1-13]. BLAST result showed that ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 sequences of NL1-7 were identical to C. gloeosporioides at a high level (>99%). The sequences of NL1-10 and NL1-13 were consistent with C. siamense at a high level (>99%). A maximum likelihood and Bayesian Inference analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2) placed NL1-7 in the clade of C. gloeosporioides sensu stricto and NL1-10 and NL1-13 in the clade of C. siamense. To confirm their pathogenicity, 9 healthy 3-yr-old seedlings, and 10 leaves/seedling were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (106 conidia/mL) of the 3 isolates, respectively. Three control plants were treated with sterile water. Seedlings were covered with plastic bags after inoculation and kept in a greenhouse at 25 ± 2°C and RH 80%. Within 7 days, all inoculated leaves showed lesions similar to those in the field, and controls were asymptomatic. C. gloeosporioides s.s. and C. siamense were reisolated from the infected tissues. It was reported that Colletotrichum species can cause many plant diseases, for example, C. acutatum causes twig canker (Swain et al. 2012), and C. salicis causes willow anthracnose (Okorski et al. 2018), etc. However, some Colletotrichum species are endophytic (Martin et al. 2021) and may only become pathogenic under the right conditions. This is the first report of C. gloeosporioides s.s. and C. siamense causing leaf spots on S. babylonica in the world. These data will help select appropriate strategies for managing this disease and further studies on the pathogen and the host.

First Report of Erysiphe magnoliicola Causing Powdery Mildew of Magnolia × soulangeana in China.

Magnolia × soulangeana Soul.-Bod., the saucer magnolia is an important woody ornamental plant cultivated widely in China, UK and USA. In August 2021, symptoms and signs of powdery mildew appeared on leaves of M. × soulangeana at the campus of Nanjing Forestry University (NJFU). The powdery mildew mainly infected young seedlings, with an incidence of 96.8% (436/450 seedlings), and some adult trees also been infected (5/30 trees). The mycelium was amphigenous, thinly effused or conspicuous, forming circular to irregular white patches. Noticeable brown lesions and necrosis occurred in the later stage of infection. Chasmothecia started to develop in October, 2021 and fully matured in early November, 2021. Ten fresh specimens were collected and examined to identify of the pathogen. Photos were taken with a ZEISS Axio Imager A2m microscope, a Zeiss stereo microscope (SteRo Discovery v20), and a scanning electronic microscope (JSM-7600F). Conidiophores arose from the upper part of mother cells, 78.5 ± 11.2× 10.9 ± 1.7 µm (n=30). Foot cells in conidiophores are straight and cylindrical with a constricted basal septum close to hyphal mother cell, 33.6 ± 4.3 × 10.3 ± 1.2 µm (n=30). Conidia were hyaline, ellipsoid to oval, solitary or in chains of two to four, 38.5 ± 3.3 × 18.4 ± 1.0 µm (n=30). Chasmothecia were amphigenous, scattered or aggregated, blackish brown, oblate, 101.1 ± 11.4µm diam. (n=30), with 6-10 appendages. Appendages were aseptate, rarely 1-septate, 5-6 times frequently dichotomously branched; tips were noticeably recurved, brown at the base, 105.1 ± 10.7 × 8.5 ± 1.4 µm (n=30). Asci were 6 to 8 per chasmothecium (n=30), ellipsoid to obovoid or saccate with a short stalk or sessile, 64.2 ± 6.5× 46.1 ± 5.7 um (n=30) in length, 4 to 6 spored. Ascospores were oblong-ovoid, 26.2 ± 1.4 × 13.8 ± 0.7 µm (n=30). Based on the morphological characteristics, the fungus was identified as Erysiphe magnoliicola S.E. Cho, S. Takam. & H.D. Shin. To confirm the causal fungus identity, a representative voucher specimen collected and deposited in herbarium of NJFU (NF50000008) was used for a phylogenetic analysis. Mycelia and conidia were collected from diseased leaves and genomic DNA of the pathogen was extracted. The internal transcribed spacer region (ITS) and large subunit (LSU) loci were amplified with primers ITS1/ITS4 and LR0R/LR05. The resulting sequences were deposited in GenBank (OL454094 for ITS, OM758416 for LSU). BLAST results showed that the ITS sequence was highly similar with a sequence of E. magnoliicola (type) [KJ567072, 614/619 (99.2%)], while LSU sequence was highly similar with E. magnoliicola [KJ567068, 889/891 (99.8%)] and E. magnoliae [JX235969, 903/909 (99.3%)]. Phylogenetic analyses using ITS and LSU sequences with maximum likelihood and Bayesian posterior probability using IQ-TREE v. 1.6.8 and MrBayes v. 3.2.6 placed this fungus in the E. magnoliicola clade. Based on the morphology and phylogeny, the fungus was identified as E. magnoliicola. Pathogenicity tests were carried out on six potted plants of M. × soulangeana. Three seedlings were inoculated by gently pressing the naturally infected leaves onto healthy leaves. Healthy leaves from three other seedlings served as control. Inoculated and control seedlings were placed in separate growth chambers at 23 ± 2°C/16 ± 2°C, 70% relative humidity, with a 16 h/8 h light/dark period. Symptoms developed 10 days after inoculation. The powdery mildew developing on the inoculated seedlings was examined, sequenced and confirmed as E. magnoliicola. The control leaves did not develop powdery mildew. Magnolia × soulangeana is a hybrid of Magnolia denudata × Magnolia liliiflora, both species, as well as M. sieboldii were already known as host plants of E. magnoliicola. This is the first report of powdery mildew caused by E. magnoliicola on M. × soulangeana. This finding provides crucial information for developing effective strategies to monitor and manage this disease.

First Report of Top Blight of Cunninghamia lanceolata Caused by Diaporthe unshiuensis and Diaporthe hongkongensis in China.

Cunninghamia lanceolata (Lamb.) Hook. is an important conifer species widely planted in southern China. A top blight, with an incidence of 20% (40/200 seedlings), occurred on 1-year-old seedlings of C. lanceolata in a nursery, Luzhai, Guangxi, China in August 2021. The disease mainly occurred on shoot tips. The infected needles and shoots appeared brown to brownish red. White conidial tendrils oozed from pycnidia under wet-weather conditions. Lesion margins from fresh samples were cut into small pieces (n=100), which were sterilized according to Mao et al., and placed on potato dextrose agar (PDA) at 25°C. Three isolates (GXJ2, GXJ4, and GXJ6) were obtained and deposited in The China Forestry Culture Collection Center (CFCC 55717, CFCC 55716, and CFCC 55722). The colony of GXJ2 on PDA was white, with sparse aerial mycelia, and became grey with time. The α conidia were fusiform, hyaline, and aseptate, 6.7±0.6 µm × 2.6±0.2 µm (n=30). The ß conidia were filiform, hyaline, and curved, 30.4±2.1 µm × 1.4±0.1 µm (n=30). Colonies of GXJ4 and GXJ6 were white, with moderate aerial mycelia, which collapsed at the center, and the collapsed parts were iron-gray. The α conidia were 7.8±0.8 µm × 2.5±0.2 µm (n=30). The ß conidia were absent. Morphological characters of 3 isolates matched those of Diaporthe spp.. The partial sequences of ITS, EF1-α, CAL, ß-tub, and HIS genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R and CAL228F/CAL737R, ßt2a/ßt2b and CYLH3F/H3-1b according to Gomes et al. 2013, respectively. The sequences for the five genes of each of 3 isolates were deposited in GenBank (Accession Nos. see Supplementary Table 1). BLAST results showed that the ITS, EF1-α, ß-tub, HIS, and CAL sequences of GXJ2 were highly similar (>99%) with sequences of Diaporthe unshiuensis, while sequences of GXJ4 and GXJ6 were highly similar (>99%) to those of D. hongkongensis (Supplementary Table 1). Phylogenetic analyses using concatenated sequences placed GXJ2 in the clade of D. unshiuensis, while GXJ4 and GXJ6 in the clade of D. hongkongensis. Based on the phylogeny and morphology, GXJ2 was identified as D. unshiuensis, GXJ4 and GXJ6 as D. hongkongensis. Pathogenicity tests were performed on nine 1-year-old seedlings of C. lanceolata, and 10 needles at shoot tip per seedling were slightly wounded and inoculated with 5-mm mycelial plugs from one of 3 isolates. Three control seedlings were treated with PDA plugs. Each plant was covered with a plastic bag after inoculation and kept in an air-conditioned nursery at 25°C/16°C (day/night). The symptoms appeared 5-8 days after inoculation and were similar to those observed in the nursery. D. unshiuensis and D. hongkongensis were re-isolated from the inoculated seedlings and were confirmed based on morphology and ITS sequences. The controls were symptomless, and no fungus was isolated from them. D. unshiuensis was first reported as an endophyte on the fruit of Citrus unshiu, and caused peach constriction canker, shoot blight of kiwifruit. D. hongkongensis was first described from fruit of Dichroa febrifuga and caused shoot canker of pear, shoot blight and leaf spot of kiwifruit, and fruit rot of peach. This is the first report of D. unshiuensis and D. hongkongensis causing the top blight of C. lanceolata. This study provides a basis for controlling this newly emerging disease in the nursery.

Red Foliage Blight of × Taxodiomeria peizhongii Caused by Neopestalotiopsis clavispora Newly Reported in China.

× Taxodiomeria peizhongii Z.J. Ye, J.J. Zhang & S.H. Pan is a hybrid of Taxodium mucronatum and Cryptomeria fortunei. It can adapt to various site conditions and has a good saline-alkali tolerance, which is a unique tree species in eastern China. In August 2020, a red foliage blight with an incidence of 70% (105/150 plants) was found on the leaves of × T. peizhongii in a nursery, Shanghai, China (121°21'12"E, 31°41'56"N). It developed from apical leaves of branches downwards. The infected leaves became reddish brown and withered. Fresh specimens were collected from 3 infected trees. Small samples (3-4 mm2) from lesion margins were sterilized, plated on potato dextrose agar (PDA) and incubated at 25°C. Nine isolates of the same fungus were obtained. Three representative isolates (DFS1-3, DFS1-8, and DFS1-9) were used for morphological and molecular studies and deposited in the China's Forestry Culture Collection Center (cfcc57401 to cfcc57403). The colonies of three isolates on PDA grew fast, covering the entire plate with white cottony mycelia in 7 days. Acervuli of DFS1-3 were 618-996 × 586-945 µm (n = 50). Conidiogenous cells were 4.4-9.8 µm (n = 50) long. Conidia were 5-celled, clavate to fusiform, smooth, 19-24 × 6.4-8.8 µm (n = 50). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4-8 µm long; n = 50), and 2-3 apical appendages (15-30 µm long; n = 50), filiform. The morphological characters of DFS1-8 and DFS1-9 were almost identical to DFS1-3. Based on morphological studies, the isolates were Neopestalotiopsis sp.. The DNA of 3 isolates was extracted. The internal transcribed spacer region (ITS), ß-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) loci were amplified using the primer pairs ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2. BLAST result showed that ITS of the three isolates were identical to Pestalotiopsis sp. at a high level (greater than 99%), and TUB2, TEF1-α were highly similar with Neopestalotiopsis sp. (greater than 99%). The sequences were deposited in GenBank [Accession Nos. OM188301 and OM222696 to OM222697 for DFS1-3; OM188303 and OM222698 to OM222699 for DFS1-8; OM188302 and OM222700 to OM222701 for DFS1-9]. A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, TUB2, TEF1-α) clustered 3 isolates together with N. clavispora including type isolate (MFLUCC 12-0281). Based on the morphology and phylogeny, the fungus was N. clavispora. To confirm pathogenicity, 9 healthy 2-yr-old seedlings, and 10 leaves per seedling were wounded with a sterile needle and inoculated with conidial suspension (106 conidia/mL). Three control plants were sprayed with sterile water. Seedlings were covered with plastic bags after inoculation and kept in a greenhouse at 25 ± 2°C and RH 80%. Seven days after inoculation, all inoculated leaves were reddish brown and withered like those observed in the field, whereas the control plants remained symptomless. N. clavispora was successfully reisolated from the infected tissues. This pathogen has been reported to cause leaf blight on many other hosts, such as Ligustrum lucidum and macadamia, but in recent years, the disease has also been reported on flowers, such as Anthurium. It has not been reported on Taxodium and Cryptomeria. This is the first report of N. clavispora infecting × T. peizhongii in the world. These data will help select appropriate fungicides for managing this newly emerging disease.

Striatibotrys neoeucylindrosporus sp. nov., a Stachybotrys-like fungus from North America.

Two isolates from Canada and the USA (UAMH 7122 and UAMH 7211, respectively) previously identified as Stachybotrys eucylindrosporus were studied by morphology and six-locus phylogeny (cmdA, ITS, LSU, rpb2, tef1α and tub2). UAMH 7122 and UAMH 7211 are morphologically related but phylogenetically distinct from Striatibotrys eucylindrosporus (≡Stachybotrys eucylindrosporus) and Str. rhabdosporus. Hence, UAMH 7122 and UAMH 7211 are described as a new species, Striatibotrys neoeucylindrosporus sp. nov. with UAMH 7211 as the holotype. The characters of this species include some phialides proliferating by holoblastic extension of phialides and conidia clavate, subcylindrical or cylindrical ellipsoid, or dumbbell-shaped, dark brown to olivaceous grey when mature, longitudinally striate, 10.3-12.3×3-3.8 µm. A key to the species of Striatibotrys is provided.

The Fungal Endophyte Epicoccum dendrobii as a Potential Biocontrol Agent Against Colletotrichum gloeosporioides.

Anthracnose caused by Colletotrichum gloeosporioides is one of most serious fungal diseases on Chinese fir (Cunninghamia lanceolata). Eight fungal endophytes were isolated from a young heathy branch of Chinese fir and screened against the pathogen in vitro. One isolate, designated as SMEL1 and subsequently identified as Epicoccum dendrobii based on morphological and phylogenetic analyses, suppressed mycelial growth of Colletotrichum gloeosporioides on dual-culture plates. Additionally, E. dendrobii metabolites significantly decreased the biomass of Colletotrichum gloeosporioides. E. dendrobii was able to enter the internal tissues of the host plant via stomatal cells. Metabolites of E. dendrobii significantly inhibited conidial germination and appressorium formation, which at least partly explained why the endophyte significantly inhibited lesion development caused by Colletotrichum gloeosporioides on various host plants. We further confirmed that some components with antifungal activity could be extracted from E. dendrobii using ethyl acetate as an organic solvent. To our knowledge, this is the first report of E. dendrobii as a potential biocontrol agent against a fungal phytopathogen.

First report of leaf spot caused by Colletotrichum siamense on Salix matsudana in China.

Salix matsudana Koidz. (Chinese willow) is an important landscaping tree species widely grown in China (Zhang et al. 2017). In October 2019, a characteristic leaf spot disease of S. matsudana was found on the campus of Nanjing Forestry University. Most 25-year-old S. matsudana trees (13 out of 21, approximately 62%) on campus showed the leaf spot disease. On average, 70% of the leaves per individual tree were affected by this disease. Foliar symptoms began as dark brown, irregular spots and the centers were gray-white, gradually enlarging with time. Leaf spot symptomatic leaves were collected from three infected S. matsudana trees (10 leaves/tree), and small infected tissues (3-4 mm2) were surface-sterilized in 75% ethanol for 30 s, 1% NaClO for 90 s, rinsed in ddH2O, dried on sterilized filter paper, and plated on potato dextrose agar (PDA), and then incubated at 25°C. Three isolates (NHY1-1, NHY1-2, and NHY1-3) of the same fungus were obtained in 85% of the samples and deposited in China's Forestry Culture Collection Center (NHY1-1: cfcc55354, NHY1-2: cfcc55355, NHY1-3: cfcc55359). The colonies of three isolates were white, but the reverse side was grayish-white. The conidia of NHY1-1 were one-celled, straight, subcylindrical, hyaline, 14.4 ± 0.9 × 5.4 ± 0.4 µm (n = 50), with a rounded end. Conidiophores were hyaline to pale brown, septate, and branched. Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled, 8.0 ± 0.9 × 5.9 ± 0.5 µm (n = 50). The conidia and appressoria of the other two isolates weralmost identical to NHY1-1. The morphological characters of the three isolates were matched with those of the Colletotrichum gloeosporioides complex (Weir et al. 2012). For accurate identification, the DNA of the three isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), superoxide dismutase (SOD2), and ß-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. MW784679 and MW808959 to MW808964 for NHY1-1; MW784726 and MW808965 to MW808970 for NHY1-2; MW784729 and MW808971 to MW808976 for NHY1-3]. A BLAST search of GenBank showed that ITS, ACT, CAL, GAPDH, SOD2, and TUB2 sequences of the three isolates were identical to Colletotrichum siamense at a high level (>99%), and CHS-1 sequences of three isolates were consistent with Colletotrichum fructicola at a high level (>99%). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, SOD2, and TUB2) placed NHY1-1, NHY1-2, and NHY1-3 in the clade of C. siamense with high bootstrap support values (ML/BI = 93/1). The pathogenicity of three isolates were tested on potted 2-yr-old seedlings (50-cm tall) of S. matsudana, which were grown in a greenhouse. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of conidial suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). In total, 12 seedlings were inoculated including controls. Three seedlings/isolate and 10 leaves/seedling were used for each treatment. The plants were covered with plastic bags after inoculation and sterilized H2O was sprayed into the bags twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2 / 16 ± 2°C. Within 7 days, all the inoculated points showed lesions similar to those observed in field, whereas controls were asymptomatic. The infection rate of each of the three isolates is 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. The diseases caused by C. siamense often occur in tropical and subtropical regions of China, with a wide range of hosts, such as Hevea brasiliensis and Coffea arabica, etc. (Cao et al. 2019; Liu et al. 2018). This is the first report of C. siamense causing leaf spot of S. matsudana in China and the world. These data will help to develop effective strategies for managing this newly emerging disease.

The Identification of a New Species, Diaporthe humulicola, a Pathogen Causing Diaporthe Leaf Spot on Common Hop.

Common hop, Humulus lupulus, is a commercially important crop in the United States, with an increasing number of hop yards being established in the Northeast. In 2018, a new fungal disease was observed at two research hop yards in Connecticut. This new pathogen affected all hop cultivars being grown and caused leaf spots and browning of cones. The causal organism was isolated and Koch's postulates were performed to confirm pathogenicity. The disease symptoms were similar to the previously described Phoma wilt; however, morphological and phylogenetic analyses placed the causal organism as a new species of Diaporthe. We propose the name Diaporthe humulicola. The disease increased under hot, humid conditions (around 24°C and 90% relative humidity), which prevail during the summer in the northeastern United States as well as other parts of the country. An in vitro preliminary assessment of fungicide sensitivity revealed that pyraclostrobin and boscalid inhibited D. humulicola growth in culture and should be further assessed for field efficacy against this new disease of hop. The proper identification and monitoring of this pathogen will be important to inform hop growers of this new threat.