First Report of Colletotrichum theobromicola Causing Anthracnose on Euonymus japonicus in China.

Euonymus japonicus, a broad-leaved evergreen tree, is widely planted in the parks and landscapes in the world (Huang et al. 2016). In 2019, anthracnose lesions were found on leaves of E. japonicus with 4~15% incidence from the Wufulinglong park (116°28' E, 39°94' N) in the Haidian District of Beijing, China. Irregular chlorotic spots appeared on the surface of the leaves, then larger necrotic spots with numerous acervuli gradually formed, and finally the infected leaves dried and dropped, detracting from the aesthetic of the landscape. To identify the pathogen, six representative leaves with typical symptoms in Wufulinglong park from three plants were collected and diseased tissue was cut into 2 mm × 2 mm pieces, disinfested in 75% ethanol for 20 s, rinsed twice in sterile water, plated onto potato dextrose agar (PDA), which were then incubated at 25℃ under 12 h photoperiod. A total of 24 morphologically identical isolates were obtained from the samples. Two of these isolates were randomly selected for further analysis to confirm their identity. The cultures (HDwfll1907HY and HDwfll1908HY) were deposited in the culture collection of the Beijing Academy of Agriculture and Forestry Sciences, China. Furthermore, genomic DNA was extracted and the sequences of internal transcribed spacer (ITS) regions, calmodulin (CAL), beta tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS) were amplified using the primers ITS1/ITS4 (Gardes et al. 1993, White et al. 1990), CL1C/CL2C (Weir et al. 2012), T1/Bt2b (Glass et al. 1995, O'Donnell et al. 1997), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/783R and CHS-79F/345R (Carbone et al. 1999), respectively. Newly obtained sequences were deposited into GenBank with accession numbers MZ229612~13 and MZ305422~31. Phylogenetic tree based on the assessed gene loci revealed that both strains clustered closely with C. theobromicola. The strain HDwfll1907HY was randomly selected for morphological description and pathogenicity assay. The colony was initially light gray with dense aerial mycelia on PDA, which later turn to gray with grayish green on the reverse side of plates. Conidiogenous cells were cylindrical, arising from swollen hyphae. Conidia were single-celled, hyaline, subcylindrical to clavate, often with broadly rounded ends, 13.78-21.99×3.47-5.44 µm (n=50). Both phylogenetic analysis and morphology support the identification of two strains as C. theobromicola. Pathogenicity tests were performed on three healthy one-year-old E. japonicus plants using the randomly selected isolate HDwfll1907HY. The leaves were sprayed with 20 mL of conidial suspension (107 conidia/mL), sterilized water inoculation under the same condition was used as control. All the treated plants were incubated in the incubator at 25°C and 90% relative humidity under 12 h photoperiod. After 18 d, all the leaves treated with the conidial suspension showed typical symptoms of anthracnose, similar to those in the field, whereas the control leaves remained symptomless. The disease assay was replicated three times for consistency. The same fungus was reisolated from the infected leaves and identified as C. theobromicola. This is the first report of C. theobromicola causing anthracnose on E. japonicus, which provides the foundation for the management of anthracnose on E. japonicus.

First report of leaf blight caused by Alternaria alternata on Platanus acerifolia in China.

Platanus acerifolia Willd. is widely planted in cities in China due to its strong adaptability to different environmental conditions. In August 2021, light brown, oval to circular, sunken spots were observed on leaves of P. acerifolia trees with 8-35% incidence, leading to severe necrosis and abscission of leaves on a street in Haidian district of Beijing (116°29'84''E, 39°95'93''N). Small pieces (5 mm×5 mm) were taken from the margin of diseased tissues, disinfected with 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed with sterile water, then plated on potato dextrose agar (PDA) and incubated at 28°C. After 4 days, representative isolates were transferred to new PDA plates. Four isolates with similar morphological characteristics were obtained and deposited in the culture collection (ID: DAA3, DAA5, DAA6 and DAA7) of our laboratory. Colonies on PDA were dense, fluffy, and light to dark gray, with a prominent white margin. Conidia formed in chains on the branched conidiophores, and were obpyriform to ellipsoid, 19.5-32.3×5.5-10.2 µm (average=26.4×7.1 µm, n=30) in size, with 3 to 5 transversal and 1 to 3 longitudinal septa. These morphological characteristics matched those of Alternaria spp. (Simmons 2007). Genomic DNA was extracted with modified CTAB method and the internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB2), glyceraldehyde 3 - dehydrogenase (GPD), endopolygalacturonase (EndoPG), as well as Alternaria major allergen (Alt a1) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), gpd1/gpd2 (Berbee et al. 1999), PG3/PG2b (Andrew et al. 2009), and Alt-for/Alt-rev (Hong et al. 2005), respectively. The obtained sequences were deposited in GenBank (accession numbers: OM228653-OM228656, OM221523-OM221542). In a BLAST search, the sequences were 100% identical with corresponding sequences of A. alternata. Phylogenetic analysis based on combined sequences using maximum parsimony method showed that the four isolates clustered together with the type strain CBS 916.96 of A. alternata. For pathogenicity test, three healthy leaves of three one-year-old P. acerifolia plants were wounded with a sterile needle and inoculated with 20 µl of spore suspension (106 conidia/ml). Plants inoculated with sterile water were treated as control. The pathogenicity test was also conducted on the unwounded leaves. After 8 days of inoculation at 25°C and 90% RH with a 12-h photoperiod, the symptoms on spore suspension-inoculated leaves were similar to those observed on trees in the street, whereas the control leaves remained symptomless. Lesions on wounded leaves were larger than those on unwounded leaves. The assay was repeated twice with consistent results. The pathogen was re-isolated from symptomatic leaf tissues and identified based on morphological and rDNA-ITS sequencing, thus, fulfilling Koch's postulates. Examples of other tree species where Alternaria alternata has been reported to cause leaf blight were Ophiopogon japonicas in China (Wang et al. 2021) and Pistacia terebinthus in Spain (López-Moral et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf blight on P. acerifolia in China. The identification could provide information for developing effective disease management strategies.

First report of Diaporthe eres causing leaf spot on Platanus acerifolia in China.

Platanus acerifolia Willd. is one of the world famous urban greening trees, known as "the king of street trees" (Loretta et al. 2020). In August 2021, severe leaf spot disease was observed on P. acerifolia with 15% incidence on a street of Haidian district (116°29'E, 39°95'N), Beijing municipality, China. Typical symptoms were small and irregular brown spots with or without yellow haloes, which gradually expanded, coalesced and became necrotic lesions. For pathogen isolation, the leaf lesions were cut into small tissue pieces, disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated on potato dextrose agar (PDA) plates. After incubation at 28°C for 4 days, three fungal isolates (FTDX2, FTDX3, and FTDX6) with similar colony characteristics were obtained after single spore isolation. Colonies were white with fluffy aerial mycelia, abundant pycnidia with black stomata appeared and cream-white liquid oozed after 20 days. Alpha conidia were 7.9 ± 0.6 × 2.5 ± 0.3 µm (n = 30), aseptate, hyaline, fusiform to ellipsoidal, and often biguttulate, while beta conidia were 22.7 ± 1.3 × 1.1 ± 0.1 µm (n = 30), aseptate, hyaline, linear, curved or hamate. The morphological characteristics were consistent with those of Diaporthe sp. (Udayanga et al. 2014). For further identification, total DNA was extracted from the three isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α), beta-tubulin (TUB), calmodulin (CAL) and histone (HIS) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT2a/BT2b (Glass and Donaldson 1995), CL1/CL2A (O'Donnell et al. 2000) and CYLH3F/H3-1b (Crous et al. 2014), respectively. The sequences were all deposited in GenBank (accession nos. OL870615 - OL870617 for ITS, OL870618 - OL870620 for EF1-α, OL870621 - OL870623 for TUB, OL870624 - OL870626 for CAL, and OL870627 - OL870629 for HIS) and aligned using BLASTn, obtaining 99-100% homology with the corresponding sequences of known Diaporthe eres strains in NCBI. Phylogenetic analysis of the combined sequences attributed the three isolates to the Diaporthe eres clade. Pathogenicity tests were performed on three healthy one-year-old P. acerifolia plants using the randomly selected isolate FTDX2. The leaves were inoculated with 20 µl of spore suspension (106 conidia/ml), with or without wound pretreatment, sterilized water inoculation under the same condition was used as control. All the treated plants were incubated in the greenhouse at 25°C and 90% RH with a 12-h photoperiod. After 8 days, the inoculated plants showed spot symptoms on leaves similar to those previously observed, whereas the control leaves remained symptomless. Lesions on the wounded leaves were much larger in size compared with those unwounded. The same pathogen was re-isolated and identified based on morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Diaporthe eres has been reported to cause leaf spot on many horticultural plants, such as Photinia fraseri (Song et al. 2019) and Podocarpus macrophyllus (Zheng et al. 2020). To our knowledge, this is the first report of D. eres causing leaf spot on Platanus acerifolia in China. This finding is a valuable contribution to the knowledge on leaf spot disease development in horticultural plants.

MiR-125b Improves acute myocardial infarction in rats by regulating P38/Sirtl/P53 signaling pathway.

The aim of this study was to investigate the differential expression of micro ribonucleic acid (miR)- 125b in acute myocardial infarction (AMI) cases, and to explore the mechanism by which it affects cardiac function. Sprague-Dawley rats were used for AMI modeling, and the expression of miR-125b in the myocardial tissues of AMI rats was detected via fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Thereafter, the target genes of miR-125b were collected and uploaded to WenGestalt for gene ontology (GO) and pathway enrichment analyses. In-vitro experiments were then applied to determine the p38-sirtuin 1 (Sirt1)-p53 expression change and cardiomyocyte apoptosis under down-regulation of miR-125b. Next, the interaction between miR-125b and its target genes was verified by luciferase reporter gene assay. The expression of miR-125b in the cardiac tissues was decreased in theAMI group compared with that in the Sham group (p<0.05). The luciferase reporter gene assay confirmed that p38 was the target gene of miR-125b. Furthermore, the down-regulated expression of miR-125b in H9C2 cells up-regulated the protein expressions of p38 and phosphorylated p38, thus activating the Sirt1-p53 signaling pathway. Moreover, the down-regulation of miR-125b expression in H9C2 cells gave rise to the elevated apoptosis rate, and the down-regulated expression of miR-125b induced cardiomyocyte apoptosis through activating the p38-Sirt1-p53 signaling pathway.

A comparative study at two different altitudes with two dietary nutrition levels on rumen fermentation and energy metabolism in Chinese Holstein cows.

The object of this study was to investigate the effect of two altitudes (1600 vs. 3600 m) with two nutritional levels [5.88 MJ/kg dry matter (DM) vs. 7.56 MJ/kg DM] on apparent total tract digestibility, rumen fermentation, energy metabolism, milk yield and milk composition in Chinese Holstein cows. Sixteen Chinese Holstein cows in their third lactation with close body weights, days in milk and milk yield were randomly divided into four groups, of which two were directly transferred from Lanzhou (altitude of 1600 m) to Lhasa (altitude of 3600 m). Four treatments (high plateau and high nutrition level, HA-HN; high plateau and low nutrition level, HA-LN; low plateau and high nutrition level, LA-HN; and low plateau and low nutrition level, LA-LN) were randomly arranged in a 2 × 2 factorial experimental design. Results indicated that the apparent total tract digestibility of a diet's DM, organic matter, crude protein, neutral detergent fibre and acid detergent fibre and DM intake were not affected by either altitude or nutrition level (p > 0.05). Milk protein percentage was higher for the diet with the high level of nutrition than for the diet with low nutrition level irrespective of altitude (p < 0.05). Percentages of milk fat and milk lactose were not affected by either altitude or nutrition level (p > 0.05). The metabolizable energy used for milk energy output was decreased by high altitude in comparison with that at low altitude (p < 0.05). No differences were observed in the live body weight or body condition score (BCS) of Chinese Holstein cows among all of the four treatments (p > 0.05).

Effect of several supplemental Chinese herbs additives on rumen fermentation, antioxidant function and nutrient digestibility in sheep.

Two experiments were carried out in this study. Experiment 1 was conducted to examine the effects of several supplemental Chinese herbs on antioxidant function and slaughtered body weight in sheep. Results indicated that Fructus Ligustri Lucidi supplementation improved the blood antioxidant function [higher concentration of glutathione reductase (GR), superoxide dismutase and lower concentration of malondialdehyde] and slaughtered body weight in sheep (p < 0.05). Experiment 2 was conducted to investigate the effect of Fructus Ligustri Lucidi extract (FLLE) on rumen fermentation and nutrient digestibility in sheep. Four levels of FLLE treatments, i.e. 0, 100, 300 and 500 mg/kg dry matter (DM), were used in this part. Addition of FLLE at 300 or 500 mg/kg DM increased total volatile fatty acid (VFA) concentration and propionate proportion, decreased ammonia-N concentration in the ruminal fluid, reduced blood urea nitrogen concentration at 2, 4, 6 and 8 h after morning feeding (p < 0.05). Addition of FLLE at all dosages had no effect on ruminal pH value and acetate concentration at all sampling time points in sheep (p > 0.05). Dynamic degradation coefficient c of maize DM was significantly increased by supplementing FLLE at 300 or 500 mg/kg DM (p < 0.05). Fructus Ligustri Lucidi extract addition had no effect on degradation coefficients a, b, c of DM and nitrogen of soybean meal; a, b of maize DM; a, b, c of maize nitrogen; and a, b, c of neutral detergent fibre (NDF) and acid detergent fibre (ADF) of Chinese wildrye (p > 0.05). Addition of FLLE at 300 or 500 mg/kg DM increased DM and organic matter digestibility of diet (p < 0.05). Fructus Ligustri Lucidi extract addition had no effect on digestibility of diet's NDF, ADF and crude protein (p > 0.05). From the aforementioned results, it is indicated that FLLE improved antioxidant status and slaughtered body weight. Fructus Ligustri Lucidi extract addition has capability to modulate rumen fermentation, increase the maize degradation rate, total volatile fatty acid concentration and propionate proportion in sheep.

Effect of supplemental Bacillus cultures on rumen fermentation and milk yield in Chinese Holstein cows.

Two experiments were conducted to study the effect of supplemental 100 g/day of live Bacillus cultures (2 x 10(11) cell of Bacillus subtilis and Bacillus licheniformis) on rumen fermentation as well as milk yield and composition in Chinese Holstein cows. In experiment 1, investigating 3 x 10 cows, milk yield and milk protein were increased by using B. licheniformis (p < 0.05) in comparison with an unsupplemented group and the B. subtilis group. Body weight was not significantly affected by Bacillus culture supplementation (p > 0.05). Percentage of milk fat and lactose was not significantly different between treatments (p > 0.05). But milk protein increased with B. licheniformis supplementation (p < 0.05). In experiment 2, carried out with three non-lactating ruminally and duodenally fistulated cows, results showed that B. licheniformis supplementation increased microbial crude protein flow into duodenum (p < 0.05) and decreased the ammonia nitrogen concentration in ruminal fluid at 0.5 h, 1 h, 3 h, 6 h after morning feeding (p < 0.05). Bacillus licheniformis supplementation increased total VFA and acetate concentration in ruminal fluid at 0.5 h, 1 h, 3 h, 6 h after morning feeding (p < 0.05). Bacillus subtilis had no significant effect on rumen fermentation characteristics, duodenal microbial N flow and ruminal apparent nutrient digestibility (p > 0.05). Bacillus licheniformis increased ruminal apparent nutrient digestibility of neutral detergent fibre, acid detergent fibre, and organic matter (p < 0.05).