Description of two new Megistophylla Burmeister, 1855 species from China, with a redescription of M. andrewesi Moser, 1913 (Coleoptera: Scarabaeidae: Melolonthinae).

This study presents descriptions and illustrations of two new melolonthine species: Megistophylla brevivirgata Wang & Gao, new species and M. keithi Wang & Gao, new species from Yunnan province in China. Additionally, a detailed redescription and illustration of the male of M. andrewesi Moser, 1913, and a description of its previously unknown female are provided. Finally, a key to identification of Megistophylla Burmeister, 1855 species from China is added.

First Report of Colletotrichum godetiae Causing Anthracnose on Walnut (Juglans regia and Juglans sigillata) in China.

In August 2020, anthracnose lesions were observed on fruits of Juglans regia and J. sigillata in walnut orchards, in Yijun (Shaanxi Province) and Nanhua (Yunnan Province) counties, China. Symptoms on walnut fruits first appeared as small necrotic spots that rapidly enlarged into subcircular or irregular sunken black lesions (Fig. 1a, b). Sixty diseased walnut fruits (30 fruits of J. regia and J. sigillata, respectively) were randomly sampled from six orchards (10-15 ha each orchard, three orchards were selected in each county) with severe anthracnose (incidence rate of fruit anthracnose is over 60% in the orchard.) in two counties. Twenty-six single spore isolates were obtained from diseased fruits as described by Cai et al. (2009). After seven days, isolates formed grey to milky white colony with abundant aerial hyphae on the upper surface of colony, and milky white to light olive on the back of PDA (Fig. 1c). Conidiogenous cells were hyaline, smooth-walled, and cylindrical to clavate (Fig. 1d). Conidia were smooth-walled, aseptate, cylindrical to fusiform, with both ends acute or one end round and one end slightly acute (Fig. 1e), and ranged in size from 15.5-24.3×4.9-8.1 µm (n=30). Appressoria were brown to medium brown, clavate to elliptical, with the edge entire or undulate (Fig. 1f), and ranged in size from 8.0-27.6×4.7-13.7µm (n=30). The morphological characteristics of 26 isolates were similar to those of the species complex Colletotrichum acutatum (Damm et al. 2012). Six representative isolates were randomly selected (three isolates for each province) for molecular analysis. The ribosomal internal transcribed spacers (ITS) (White et al. 1990), beta-tubulin (TUB2) (Glass and Donaldson 1995), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al.1992) and chitin synthase 1 (CHS-1) (Carbone and Kohn 1999) genes were amplified and sequenced. Sequences of 6 of 26 isolates were submitted to GenBank (Accession Nos: ITS: MT799938-MT799943, TUB: MT816321-MT816326, GAPDH:MT816327-MT816332, CHS-1: MT816333-816338). Multi-locus phylogenetic analyses revealed that six isolates clustered together with Colletotrichum godetiae ex-type culture isolates CBS133.44 and CBS130251, and the bootstrap support value was 100% (Fig.2). The pathogenicity of two representative isolates (CFCC54247 and CFCC54244) was tested using healthy fruits of the " J. regia cv. Xiangling" and " J. sigillata cv. Yangbi" varieties. Forty sterilized fruits (20 fruits were inoculated with CFCC54247, and 20 fruits with CFCC54244) were wounded by puncturing with a sterile needle through walnut pericarp and inoculated in the wound site with 10 µl of conidial suspension (106 conidia/ml) from seven day old colonies grown on PDA at 25℃. Twenty wounded fruits were inoculated with sterile water as control. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12h light/dark cycle. The experiment was repeated three times. Anthracnose symptoms (Fig. 1g-h) were observed in all inoculated fruits after 12 days, whereas controls showed no symptoms. Fungal isolates from inoculated diseased fruits showed the same morphological and molecular characteristics as the isolates obtained in this study, confirming Koch's postulates. To our knowledge, this is the first report of C. godetiae causing anthracnose on the two walnut species in China. The result will be helpful for providing a basis for further research on the control of the disease.

Meroterpenoids with Immunosuppressive Activity from Edible Fungus Craterellus odoratus.

Two unusual polyketide-sesquiterpene metabolites, craterodoratins T (1) and U (2), along with the known compound craterellin A (3), were isolated from the higher fungus Craterellus odoratus. The structures of isolated compounds were characterized based on nuclear magnetic resonance (NMR) and mass spectrum (MS) spectroscopic analysis, while the absolute configuration of the compounds was determined by theoretical NMR and electronic circular dichroism (ECD) calculations. Compound 1 possessed a rare structure with two aromatic groups. Compounds 1 and 3 showed immunosuppressive activity with IC50 values ranging from 5.516 to 19.953 µM.

Diterpenes Specially Produced by Fungi: Structures, Biological Activities, and Biosynthesis (2010-2020).

Fungi have traditionally been a very rewarding source of biologically active natural products, while diterpenoids from fungi, such as the cyathane-type diterpenoids from Cyathus and Hericium sp., the fusicoccane-type diterpenoids from Fusicoccum and Alternaria sp., the guanacastane-type diterpenoids from Coprinus and Cercospora sp., and the harziene-type diterpenoids from Trichoderma sp., often represent unique carbon skeletons as well as diverse biological functions. The abundances of novel skeletons, biological activities, and biosynthetic pathways present new opportunities for drug discovery, genome mining, and enzymology. In addition, diterpenoids peculiar to fungi also reveal the possibility of differing biological evolution, although they have similar biosynthetic pathways. In this review, we provide an overview about the structures, biological activities, evolution, organic synthesis, and biosynthesis of diterpenoids that have been specially produced by fungi from 2010 to 2020. We hope this review provides timely illumination and beneficial guidance for future research works of scholars who are interested in this area.

Sesquiterpenoids Specially Produced by Fungi: Structures, Biological Activities, Chemical and Biosynthesis (2015-2020).

Fungi are widely distributed in the terrestrial environment, freshwater, and marine habitat. Only approximately 100,000 of these have been classified although there are about 5.1 million characteristic fungi all over the world. These eukaryotic microbes produce specialized metabolites and participate in a variety of ecological functions, such as quorum detection, chemical defense, allelopathy, and maintenance of symbiosis. Fungi therefore remain an important resource for the screening and discovery of biologically active natural products. Sesquiterpenoids are arguably the richest natural products from plants and micro-organisms. The rearrangement of the 15 high-ductility carbons gave rise to a large number of different skeletons. At the same time, abundant structural variations lead to a diversification of biological activity. This review examines the isolation, structural determination, bioactivities, and synthesis of sesquiterpenoids that were specially produced by fungi over the past five years (2015-2020).

Immunosuppressive Sesquiterpenoids from the Edible Mushroom Craterellus odoratus.

The aim of this work was to comprehensively understand the chemical constituents of the edible mushroom Craterellus ordoratus and their bioactivity. A chemical investigation on this mushroom led to the isolation of 23 sesquiterpenoids including eighteen previously undescribed bergamotane sesquiterpenes, craterodoratins A-R (1-18), and one new victoxinine derivative, craterodoratin S (19). The new structures were elucidated by detailed interpretation of spectrometric data, theoretical nuclear magnetic resonance (NMR) and electronic circular dichroism (ECD) calculations, and single-crystal X-ray crystallographic analysis. Compounds 1 and 2 possess a ring-rearranged carbon skeleton. Compounds 3, 10, 12-15, 19, 20 and 23 exhibit potent inhibitory activity against the lipopolysaccharide (LPS)-induced proliferation of B lymphocyte cells with the IC50 values ranging from 0.67 to 22.68 µM. Compounds 17 and 20 inhibit the concanavalin A (ConA)-induced proliferation of T lymphocyte cell with IC50 values of 31.50 and 0.98 µM, respectively. It is suggested that C. ordoratus is a good source for bergamotane sesquiterpenoids, and their immunosuppressive activity was reported for the first time. This research is conducive to the further development and utilization of C. ordoratus.

Chemical constituents and their biological activities from the mushroom Pyropolyporus fomentarius.

The chemical constituents and their biological activities of the mushroom Pyropolyporus fomentarius were investigated in this study. Two previously undescribed pentacyclic lupane-type triterpenes, 3-formyloxybetulin and 3-formyloxybetulinic acid, two rare degraded ergosterols, pyropolincisterols A and B, along with ten known triterpenoids and four known ergosterols were isolated from the fruiting bodies of P. fomentarius. Their chemical structures were determined using a combination of spectroscopic analysis. Nine compounds exhibited certain cytotoxicities to human cancer cell lines, while polyporenic acid showed significant cytotoxicities to SMMC-7721 and A-549 with IC50 values less than 10 µM. Four compounds showed inhibitory activities against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages with IC50 values of 36.3, 25.1, 21.4, and 34.2 µM, respectively. The results of this assessment suggested that the lanostane triterpenoids and ergosterols in fruiting bodies of P. fomentarius played key roles in its folk usages.

Trichothecrotocins D-L, Antifungal Agents from a Potato-Associated Trichothecium crotocinigenum.

Seven new merosesquiterpenoids, trichothecrotocins D-J (1-7), two new trichothecene sesquiterpenoids, trichothecrotocins K (12) and L (13), and six known compounds (8-11, 14, and 15), were isolated from a potato-associated fungus, Trichothecium crotocinigenum. Compounds 5 and 6 were racemates which were further separated as pure enantiomers. Structures together with absolute configurations were established by extensive spectroscopic analysis, as well as quantum chemistry calculations on ECD and optical rotations. Compounds 1-4 are rare meroterpenoids featuring a seco-phenyl group, while 1 and 2 possessed a novel 6-6/5 fused ring system. Compounds 1-4, 8, 11, and 12 showed antifungal activity against four plant pathogens with MIC values of 8-128 µg/mL. It is suggested that the meroterpenoids produced by T. crotocinigenum may play an important role in the antifungal property of the fungus, thereby protecting the host plant, i.e., potato.

Illudane Sesquiterpenoids from Edible Mushroom Agrocybe salicacola and Their Bioactivities.

To comprehensively understand the chemical constituents of the edible mushroom Agrocybe salicacola and their biological functions, a phytochemical separation of the cultural broth of A. salicacola led to the isolation of four new illudane sesquiterpenoids, agrocybins H-K (1-4), along with 10 known analogues (5-14). Compounds 2-4 were racemates of which 2 and 3 were further separated into single enantiomers as 2a/2b and 3a/3b. All new structures with absolute configurations were elucidated on the basis of an extensive spectroscopic analysis and quantum chemistry calculations. Compound 1 possesses a new carbon skeleton that might be derived from the protoilludane backbone. Compounds 1, 5, 8, and 9 show a certain degree of cytotoxicity to five human cancer cell lines. Compound 1 shows a mild inhibitory effect on nitric oxide production with an IC50 value of 31.4 µM. It is concluded that A. salicacola is rich in illudin derivatives with potential bioactivity prospects, which would make A. salicacola a good material of medicine and food homology.

First Report of Colletotrichum aenigma Causing Walnut Anthracnose in China.

English/Persian walnut (Juglans regia L.) is grown as an economically valuable crop in temperate and subtropical regions. In August of 2018, serious fruit anthracnose, with brown to black circular or subcircular or irregular sunken lesions (Fig.1A), occurred on walnut trees ("Xiangling" and "lvling") in 33 ha., 23 ha. and 20 ha. orchards in Lincheng and Neiqiu county, in Xingtai, Hebei, China. Diseased fruits were observed on 41% (19,000 trees), 31% (13,300 trees) and 34% (11,400 trees) walnut trees. Diseased leaves, with circular or irregular brown to gray sunken lesions, were observed on 2% (19,000 trees), 2% (13,300 trees) and 1% (11,400 trees) walnut trees. From each orchard, 25 diseased fruits and leaves were collected, respectively. Twenty-one single spore isolates were obtained from fruits of three orchards and none from leaves as described by Cai et al. (2009). Six representative isolates 1811-1, 1811-4, 1811-7, 1811-8, 1811-11 and 1811-18, two from each orchard, were selected for further study. Colonies on PDA grew 11.8 mm d-1 at 25℃ under a 12/12 h light/dark cycle for 7 d. The upper side of colonies was milky (Fig.1 B), and reverse side was dark brown to brownish yellow. A few acervuli were observed on colonies. Conidiogenous cells were cylindrical to clavate, 10.6-29.7 × 3.1-5.3 µm (mean=21.3 × 4.0 µm, n=30) (Fig.1F). Setae were not observed. Conidia were smooth-walled, aseptate, straight or slightly distorted, cylindrical with one end slightly acute or broadly rounded ends, and 16.6-21.6 × 6.0-7.5 µm (mean=19.2 × 6.7 µm, n=30) (Fig.1 C). Appressoria were mostly irregular in outline, deeply lobed or lightly lobed, gray brown to dark brown, 8.3-16.6 × 7.1-14.5 µm (mean=12.5 × 9.7 µm, n=30) (Fig.1 D-E). Microscopic features were similar to the description of C. aenigma (Weir et al. 2012). To further identify isolates, the ribosomal internal transcribed spacers (ITS), ß-tubulin 2 (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS) and chitin synthase (CHS-1) loci of representative isolates were amplified using ITS4/ITS5, Bt2a/Bt2b, CL1/CL2, GDF1/GDR1, GSF1/GSR1 and CHS-79F/CHS-345R primers (Prihastuti et al. 2009; Carbone & Kohn 1999). Sequences of representative isolate 1811-1 were submitted to GenBank (ITS: MN893316, TUB: MN893317, CAL: MN893312, GAPDH: MN893314, GS: MN893315, CHS-1: MN893313). Maximum likehood analysis of sequences of representative isolates and reference sequences of Colletotrichum spp. from GenBank revealed that six isolates clustered together with C. aenigma ex-type culture ICMP18608, and the bootstrap value was 100% (Fig.2). Pathogenicity tests were conducted on walnut fruit as described by Wang et al. (2017, 2018) and Cai et al. (2009). 10 wounded and 10 nonwounded fruits ("Xiangling", 35 mm diameter) were inoculated with isolates 1811-1, 1811-7 and 1811-11 conidial suspension (106 spore/mL) obtained from 10 d colonies grown on PDA at 25℃, respectively. 10 wounded and 10 nonwounded fruits were inoculated with sterile water. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12 h light/dark cycle. After 10 days, necrotic lesions were observed in all inoculated fruits. The pathogen C. aenigma was reisolated from all inoculated fruits but not from control fruits. To our knowledge, this is the first report of C. aenigma causing walnut anthracnose in China. It is urgent to control walnut anthracnose caused by different species of Colletotrichum.

Chemical constituents and their cytotoxicities from mushroom Tricholoma imbricatum.

Two undescribed triterpenes, tricholimbrins A and B, three undescribed steroids, tricholimbrins C‒E, one undescribed 4-chromanone derivative, along with 27 known compounds were isolated from fruiting bodies of the mushroom Tricholoma imbricatum. Tricholimbrins A and B are two polycyclic triterpenoids with a carbon degradation, while tricholimbrin C is a ring-rearranged steroid containing an aromatic moiety that might be derived from an ergosterol. Isocyathisterol, 3ß,15α-dihydroxyl-(22E,24R)-ergosta-5,8(14),22-trien-7-one, demethylincisterol A3, and volemolide showed cytotoxicities to six human cancer cell lines. 3ß-Hydroxyl-(22E,24R)-ergosta-5,8,22-trien-7,15-dione and 3ß-hydroxyl-(22E,24R)-ergosta-5,8,22-trien-7-one showed preferable cytotoxicities against HL-60 while chaxine C and volemolide showed preferable cytotoxicities against A-549, with IC50 values less than 10 µM.

Melodinines Y1-Y4, four monoterpene indole alkaloids from Melodinus henryi.

Melodinines Y1-Y4 (1-4), four undescribed alkaloids were isolated from Melodinus henryi. Their structures were elucidated by extensive NMR, mass spectroscopic analyses, theoretical NMR and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of bisindole alkaloids possessing an eburnamine-leuconoxine combination. Compound 3 is a rare 2,3-seco pleiocarpamine type monoterpene indole alkaloid. Compound 1 showed cytotoxic activities against six human cancer cell lines with IC50 values of 0.5-15.2 µM.

Monoterpenoid indole alkaloids from the bark of Melodinus henryi.

Four new alkaloids, melodinines W1-W4 (1-4), together with twenty one known alkaloids (5-25) were isolated from Melodinus henryi. The structures with absolute configurations were elucidated by extensive MS and NMR spectroscopic methods, as well as the single crystal X-ray diffraction and ECD calculations. All compounds were evaluated for their cytotoxicities to five human cancer cell lines. Many compounds showed certain cytotoxicities to five human cancer cell lines with an IC50 range of 1.4-29.4 µM.

Two new and one newly recorded species of Anomala Samouelle, 1819 Coleoptera: Scarabaeidae: Rutelinae) from Yunnan, China.

Two new species, Anomala qiului Huang Wang, new species and Anomala xuhaoi Huang Wang, new species, are described and illustrated from Yunnan, China. Anomala trochanterica Arrow, 1917 is recorded from China for the first time, with the report of its intraspecific variation also discussed. Color plates (habitus and genitalia) of A. flavipunctuata Lin, 1999, A. flavoguttata Miyake, 2000, A. fuscicauda Lin, 1999 and A. trochanterica are provided for the first time.

Influence and analysis of low-dosage steroid therapy in severe aristolochic acid nephropathy patients.

AIM: To investigate the effect of low-dosage steroid therapy in patients with severe aristolochic acid nephropathy (AAN). METHODS: Forty-three chronic AAN patients in the Peking Union Medical College Hospital and the First Affiliated Hospital of Xinxiang Medical College were included in this study from November 1998 to October 2013. According to the treatment method, the patients were divided into a steroid group (SG, n = 25) and a control group (CG, n = 18). The serum biochemical indicators at the basement in the two groups exhibited no obvious statistical differences. In comparison with the baseline data, the levels of serum creatinine at 3, 6, 9, and 12 months were analyzed. The blood pressure, haemoglobin, serum biochemical indicators, and the side-effects of steroid application were also observed. Urinary macrophage chemoattractant protein-1 (MCP-1) and transforming growth factor-1 (TGF-1) amounts were measured as well. RESULTS: (i) The serum creatinine content in the CG group was significantly higher than the baseline level during the follow-up(6, 9, and 12 months later), whereas in the SG group it decreased during the 3-6 month period and remained stable within 1 year. (ii) The biochemical indicators, blood pressure, and haemoglobin persisted stable. (iii) The side-effects of low-dosage steroid therapy were not severe and were tolerated by the AAN patients. (4) Urinary MCP-1 and TGF-1 concentrations were positively correlated with serum creatinine and decreased in the SG group. CONCLUSION: Low-dosage steroid therapy reversed or delayed the renal failure progression in severe chronic AAN patients, which may be associated with the suppression of MCP-1 and TGF-ß1 activities.

VEGF suppresses epithelial-mesenchymal transition by inhibiting the expression of Smad3 and miR­192, a Smad3-dependent microRNA.

Transforming growth factor-ß1 (TGF-ß1)­induced epithelial­mesenchymal transition (EMT) is one of the important cellular and molecular mechanisms involved in renal fibrosis. Smad3 and miR-192 (a Smad3-dependent microRNA) are involved in TGF-ß1-mediated EMT. Vascular endothelial growth factor (VEGF) is a renal tubular epithelial survival factor. Therefore, in the present study, we investigated the role of Smad3 and miR­192 in the effects of VEGF on TGF­ß1­mediated tubular EMT. A human kidney cortex (HKC) cell line stably overexpressing VEGF (HKC-SOEV) was established. The normal HKC cells and HKC­SOEV cells were treated with TGF-ß1 (5 µg/l) or/and LY294002 (20 µmol/l) for 24 and 48 h (LY294002 blocks the effect of VEGF). The protein expression of Smad2, Smad3, Smad4 and phosphorylated Smad3 (p­Smad3) were measured by western blot analysis. The expression of Smad3 and miR-192 was determined by real­time PCR. E-cadherin and α-smooth muscle actin (α-SMA) expression was detected by western blot analysis and laser scanning confocal microscopy (LSCM). TGF-ß1 was found to induce the expression of α-SMA in the HKC cells. TGF-ß1 also induced Smad3, miR-192 and p-Smad3 expression, but suppressed E­cadherin expression. However, in the HKC-SOEV cells, the expression levels of α-SMA, Smad3, miR-192 and p­Smad3 upon TGF-ß1 stimulation were significantly reduced. In these cells, the suppressive effect of TGF-ß1 on E­cadherin was also reduced. Importantly, treatment with LY294002 significantly diminished the effect of VEGF. VEGF suppressed Smad3 and miR­192, and subsequently inhibited EMT induced by TGF-ß1.

VEGF ameliorates tubulointerstitial fibrosis in unilateral ureteral obstruction mice via inhibition of epithelial-mesenchymal transition.

AIM: Vascular endothelial growth factor (VEGF) has been shown to be a survival factor for renal tubular epithelial cells. In the present study, we investigated whether administration of VEGF ameliorates tubulointerstitial fibrosis in a mouse model of unilateral ureteral obstruction (UUO). METHODS: Thirty-six male CD-1 mice were randomly divided into three groups: sham-operation, UUO and UUO+VEGF group. VEGF (50 µg/kg) was subcutaneously injected twice daily from d 1 to d 14. Mice in each group were killed at d 3, 7, or 14 after the operation, and the tubulointerstitial fibrosis was histopathologically evaluated. Human proximal tubular epithelial cells (HK-2) were used for in vitro study. The expression levels of α-SMA, E-cadherin, TGF-ß1, CTGF, and BMP-7 in the kidney were determined using Western blot and RT-PCR. RESULTS: In the UUO mice, the degree of interstitial fibrosis was dramatically increased in a time-dependent manner. At d 3, 7, and 14, both the mRNA and protein expression levels for α-SMA, TGF-ß1, and CTGF were significantly upregulated, whereas those for E-cadherin and BMP-7 were significantly downregulated. At d 3 and 7, VEGF treatment significantly reduced interstitial fibrosis and the expression levels for α-SMA, TGF-ß1, and CTGF, while significantly increased the expression of E-cadherin and BMP-7, as compared with the UUO mice. At d 14 after operation, no significant differences were observed in the expression of the examined markers between VEGF-treated mice and UUO mice, with the exception of CTGF. In HK-2 cells, VEGF blocked TGF-ß1-induced α-SMA and vimentin expression and restored E-cadherin expression in a dose-dependent manner. CONCLUSION: VEGF may ameliorate renal tubulointerstitial fibrosis at the early stage in UUO mice. This effect may be related to inhibition of VEGF on renal tubular epithelial-mesenchymal transition (EMT).

Inhibition of tubulointerstitial fibrosis by pentoxifylline is associated with improvement of vascular endothelial growth factor expression.

AIM: Recent information indicates that pentoxifylline (PTX) has the ability to suppress inflammation and profibrotic cell proliferation. In this study, we investigated the effect of PTX on tubulointerstitial fibrosis and the expression of vascular endothelial growth factor (VEGF) in a rat model of obstructive nephropathy. METHODS: Wistar rats with left ureteral ligation were divided into control and PTX-treated groups. The histopathologic degree of tubulointerstitial fibrosis was scored with PAS and Masson-stained sections. The protein and mRNA for vascular endothelial growth factor (VEGF) were semiquantitatively measured with immunohistochemistry and RT-PCR. The protein for transforming growth factor beta1 (TGFbeta1) and hypoxia-induced factor 1 alpha (HIF-1alpha) was determined by Western blot. RESULTS: Compared with the control group, PTX treatment reduced fibrosis scores at d 7 and d 14 (P<0.05). The reduction was accompanied by inhibited expression of transforming growth factor-beta 1 (TGFbeta1), a key cytokine in tubulointerstitial fibrogenesis (P<0.01). Meanwhile, VEGF protein and mRNA in the kidney were increased in the PTX-treated group compared with the control group (P<0.01). PTX up-regulated expression of VEGF mRNA in a dose- and time-dependent manner in cultured HK-2 cells (P<0.01). However, expression of HIF-1alpha (a key transcription factor for VEGF gene expression) was unchanged by PTX treatment. PTX prolonged the half-life of VEGF mRNA by a 1.07-fold increase. CONCLUSIONS: PTX inhibited tubulointerstitial fibrosis in a rat model of obstructive nephropathy while preventing loss of VEGF. PTX up-regulated expression of VEGF mRNA through stabilization of its mRNA in cultured renal tubular epithelial cells.