First report of Leaf Blight of Liquidambar formosana Hance caused by Alternaria tenuissima in China.

Liquidambar formosana Hance is widely planted in urban landscapes in China owing to its ornamental red leaves. In June 2020, a distinctive leaf spot disease was observed on L. formosana in Nanjing Forestry University, Jiangsu Province of China (32°4'49"N, 118°48'56"E). Approximately 61% (14 out of 23) of the trees displayed leaf spots. The diseased symptoms included irregularly distributed spots that showed black or dark brown, and occasionally with pale green halo. Two representative trees were selected for sampling and five leaves with typical symptoms were selected randomly for isolation. The tissues from the margin of the lesions (0.2 cm × 0.2 cm) were cut and disinfected in 1% sodium hypochlorite for 90 s, rinsed in sterile water twice for 30 s, and dried with sterile paper. Then, 20 tissues were incubated on 2% potato dextrose agar (PDA) supplemented with 100 mg/L Ampicillin Sodium and incubated in the dark at 25℃ for 4 days. Seventeen single-spore fungi were isolated from lesion tissues as described by Woudenberg et al. (2013). The colony morphology of 17 isolates was extremely similar, so 3 isolates (NFUA01, NFUA02, and NFUA03) were selected randomly for further study. Colonies on PDA were circular, gray, and slightly raised loose cotton mycelium, while the reverse side was olive green in the center with white margins. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 7.1 to 32.5 × 3.3 to 13.3 µm (n=50). The morphological observations were consistent with the description of the genus Alternaria sp. (Woudenberg et al. 2013). Six gene fragments, including SSU, LSU, ITS, GAPDH, RPB2 and EF-1 region, were amplified and sequenced. The primers of six nuclear loci were used by NS1 / NS4((White et al. 1990), LSU1Fd (Crous et al. 2009)/ LR5 (Vilgalys & Hester 1990), V9G (De Hoog & Gerrits van den Ende 1998)/ ITS4 (White et al. 1990), gpd1 / gpd2 (Berbee et al. 1999), RPB2-5F2 / fRPB2-7cR (Liu et al. 1999), and EF1-728F / EF1-986R (Carbone & Kohn 1999). The sequences were submitted in GenBank (SSU, ON237470 to ON237472; LSU, ON237464 to ON237466; ITS, ON197354 to ON197356; GAPDH, ON237476 to ON237478; RPB2, ON237467 to ON237469; EF-1, ON237473 to ON237475). BLAST result showed that SSU, LSU, ITS, GAPDH, RPB2, and EF-1 sequences of NFUA01, NFUA02, and NFUA03 were identical to A. tenuissima at a high level (>99%, Table 1). A maximum likelihood and Bayesian posterior probability analysis were performed by IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (Guindon et al. 2010; Ronquist et al. 2012). The representative strains which selected for Phylogenetic analyses were chosen from the strains which mentioned by Woudenberg et al (2013) and obtained the sequences from NCBI. The concatenated sequences placed NFUA01, NFUA02 and NFUA03 in the clade of Alternaria tenuissima with a high confidence level (ML/BI= 100/1). A pathogenicity assay was done using isolate NFUA01 on 3-year-old L. formosana seedlings. L. formosana leaves were wounded by a sterilized needle (0.5-mm-diam), and inoculated with spore suspension (106 conidia/mL), and L. formosana leaves inoculated with sterile water were used as the control. Each treatment had 5 leaves, and incubated at 25℃ under high moisture conditions. The experiments were conducted three times. Seven days after inoculation, leaves inoculated with spore suspension showed brown leaf blights resembling the original disease symptoms, whereas the control remained healthy. The fungus was reisolated from the lesions and was confirmed as A. tenuissima based on morphologically characteristics and ITS sequence analysis. To our knowledge, this is the first report of A. tenuissima associated with leaf blight on L. formosana. The finding provides clear pathogen information for further evaluation of the disease control strategies.

Species of the Colletotrichum spp., the Causal Agents of Leaf Spot on European Hornbeam (Carpinus betulus).

European hornbeam (Carpinus betulus L.) is widely planted in landscaping. In October 2021 and August 2022, leaf spot was observed on C. betulus in Xuzhou, Jiangsu Province, China. To identify the causal agent of anthracnose disease on C. betulus, 23 isolates were obtained from the symptomatic leaves. Based on ITS sequences and colony morphology, these isolates were divided into four Colletotrichum groups. Koch's postulates of four Colletotrichum species showed similar symptoms observed in the field. Combining the morphological characteristics and multi-gene phylogenetic analysis of the concatenated sequences of the internal transcribed spacer (ITS) gene, Apn2-Mat1-2 intergenic spacer (ApMat) gene, the calmodulin (CAL) gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, Glutamine synthetase (GS) gene, and beta-tubulin 2 (TUB2) genes, the four Colletotrichum groups were identified as C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense. This study is the first report of four Colletotrichum species causing leaf spot on European hornbeam in China, and it provides clear pathogen information for the further evaluation of the disease control strategies.

First report of leaf spot disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China.

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.

First Report of Wilt of European hornbeam (Carpinus betulus L.) Caused by Fusarium oxysporum in China.

European hornbeam (Carpinus betulus L.) has been used as an important ornamental species for urban landscaping since the Italian Renaissance (Rocchi et al. 2010). In May 2019, 15% of 3000 C. betulus trees with wilted leaves and root rot were observed in a field (about 26 ha) in Pizhou, Jiangsu Province, China. Internal discoloration of the stem began with brown to black discoloration of the vascular system and gradually spread to inward areas. Roots and stems from symptomatic plants were washed free of soil, surface sterilized with 0.8% NaOCl, rinsed three times in sterile H2O, and blotted dry with a paper towel. Small segments (0.5-cm-long) were cut from the discolored vascular tissues, and then put on potato dextrose agar (PDA) at 25°C in darkness. After 4 days, fungal colonies were observed on the PDA. Pure cultures were obtained by monosporic isolation, and 9 morphologically similar fungal isolates (EJ-1 to EJ-9) were obtained. All purified cultures were incubated on PDA at 25°C in darkness as the initial isolation. Colonies of the 9 isolates on PDA displayed entire margins and showed abundant pink aerial mycelia initially and turned to light violet with age. Microconidia were elliptical or oval in shape, 0 septate, (5.2-)8.7(-12.5) × (3.5-)3.6(-5.5) µm. Macroconidia were falciform, 0-4 septate, and straight to slightly curved with a notched foot cell, (17.1-)20.5(-28.4) × (3.8-)4.1(-4.6) µm. These morphological characteristics resemble Fusarium oxysporum (Leslie and Summerell 2006). Genomic DNA of each isolate was extracted from mycelia using a CTAB method (Mo¨ller et al. 1992). The RPB2, TEF1 and cmdA genes were amplified and sequenced with the primers 5f2/7c (Liu et al. 2000), EF-1Ha/EF-2Tb (Carbone and Kohn 1999) and Cal228F/CAL2Rd (Groenewald et al. 2013), respectively. The sequences were deposited in GenBank (Table 1). A maximum likelihood phylogenetic analysis based on RPB2, TEF1 and cmdA sequences using MEGA7 revealed that the isolates were placed in the F. oxysporum species complex with 98% bootstrap support. Based on the morphological and molecular characters, all 9 isolates were identified as F. oxysporum. A pathogenicity experiment was conducted using 30 2-year-old C. betulus seedlings potted in sterile peat, 27 for inoculation (3 replicate plants per isolate) and 3 for a negative control. The treated plants were planted in the peat mixed with 50 ml of a conidial suspension of each isolate respectively. The negative control was inoculated with sterilized water. Conidia were harvested from colonized plates of PDA using sterilized water and adjusted to a concentration of 1×107 conidia/ml. All 30 seedlings were incubated in a greenhouse at 25°C with a relative humidity of 80% and a 12-h photoperiod. The inoculated seedlings displayed wilt symptoms within 30 to 40 days, and eventually died within 75 to 85 days after inoculation. Control plants remained symptomless. F. oxysporum was successfully reisolated from the vascular tissues of symptomatic plants, and sequences of RPB2, TEF1 and cmdA of re-isolates matched those of the original isolates. No pathogen was isolated from the tissues of control plants. The experiment was repeat twice with the similar results, fulfilling Koch's postulates. F. oxysporum is an important soil-borne pathogen and can cause disease in many economic plants, such as yellowwood (Graney et al. 2016), hickory (Zhang et al. 2015) and larch (Rolim et al. 2020). To our knowledge, this is the first report of wilt on C. betulus caused by F. oxysporum in China.

First report of leaf spot disease caused by Stemphylium eturmiunum on American sweetgum (Liquidambar styraciflua L.) 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.

Dual regulation effect of somatostatin on immunity in patients with severe sepsis caused by abdominal diseases / 中华外科杂志

<p><b>OBJECTIVE</b>to investigate the effect of somatostatin on inflammatory immune disorders and prognosis in patients with severe sepsis caused by abdominal diseases.</p><p><b>METHODS</b>fifty-three patients with severe abdominal sepsis (age > 18 years, APACHE-II score > 15) from June 2005 to June 2009 were randomly divided into Somatostatin group (n = 23) and SSC Group (n = 30). Fifteen healthy volunteers of the same age range were chosen as Control group. The SSC group was treated with classical SSC therapy, and the Somatostatin Group was treated with the same regime plus 14-peptide somatostatin continuous infusion at the dose of 6 mg/24 h for 7 days. The serum levels of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) were determined by using ELISA. CD(4)(+), CD(8)(+) T cell subsets were determined by fluorescence activated cell sorter(FACS) and CD(4)(+)/CD(8)(+) was calculated. APACHE-II score was observed on admission (d1) and day 3, 7 and 14 after treatment. Morality rates in 28 days in two groups were recorded.</p><p><b>RESULTS</b>compared with Control group, IL-10 and TNF-α levels were significantly elevated in patients with severe abdominal sepsis (P < 0.05), while CD(4)(+), CD(8)(+) T cell and CD(4)(+)/CD(8)(+) decreased significantly (P < 0.05). Compared with the Somatostatin group CD(4)(+), CD(8)(+) T cell and CD(4)(+)/CD(8)(+) on d7 and d14 in SSC Group were significantly increased (P < 0.05), while IL-10 and TNF-α decreased significantly(P < 0.05). APACHE-II scores on d3, d7, d14 of Somatostatin group were significantly lower than those of SSC group, and 28 d mortality rate also declined.</p><p><b>CONCLUSIONS</b>in patients with severe abdominal sepsis, systemic inflammatory response and immune suppression exist simultaneously. Somatostatin has a dual immunomodulatory activity in these patients.</p>