Biological control of wheat powdery mildew disease by the termite-associated fungus Aspergillus chevalieri BYST01 and potential role of secondary metabolites.

BACKGROUND: Wheat powdery mildew, caused by the biotrophic pathogen Blumeria graminis f. sp. tritici (Bgt) is a serious fungal disease. Natural metabolites produced by microorganisms are beneficial biological control agents to inhibit Bgt. In the present study, we investigated the effects of Aspergillus chevalieri BYST01 on wheat powdery mildew. RESULTS: A strain isolated from the termite was identified as A. chevalieri BYST01 by morphological characteristics and phylogenetic analysis. The fermentation broth of BYST01 showed good biocontrol effect on the Bgt in vivo with the control efficiencies of 81.59% and 71.34% under the protective and therapeutic tests, respectively. Four known metabolites, including the main compound physcion (30 mg/L), were isolated from the fermentation broth of BYST01 extracted with ethyl acetate. Importantly, under a concentration of 0.1 mM, physcion repressed conidial germination of Bgt with an inhibition rate of 77.04% in vitro and showed important control efficiencies of 80.36% and 74.64% in vivo under the protective and therapeutic tests, respectively. Hence, the BYST01 showed important potential as a microbial cell factory for the high yield of the green natural fungicide physcion. Finally, the biosynthetic gene clusters responsible for physicon production in BYST01 was predicted by analyzing a chromosome-scale genome obtained using a combination of Illumina, PacBio, and Hi-C sequencing technologies. CONCLUSION: Aspergillus chevalieri BYST01 and its main metabolite physcion had a significant control effect on wheat powdery mildew. The biosynthesis pathway of physcion in BYST01 was predicted. © 2023 Society of Chemical Industry.

Structural characterization, derivatization and antibacterial activity of secondary metabolites produced by termite-associated Streptomyces showdoensis BYF17.

BACKGROUND: Insect-associated Streptomyces is a valuable resource for development of compounds with antibacterial potential. However, relatively little is known of the secondary metabolites produced by termite-associated Streptomyces. RESULTS: Here, seven compounds including o-acetaminophenol (1), phenazine-1,6-dicarboxylic acid (2), phenylacetic acid (3), phenazinolin D (4), izumiphenazine A (5), izumiphenazine B (6) and phenazinolin E (7) were obtained from the fermentation broth of a termite-associated Streptomyces showdoensis BYF17, which was isolated from the body surfaces of Odontotermes formosanus. Two additional novel derivative compounds (6a and 6b) were synthesized via acetylation and methylation, respectively. The structures of these compounds were elucidated by spectroscopic analyses. The antibacterial bioassay showed that compound 6a displayed strong inhibitory effects against Pseudomonas syringae pv. actinidiae (Psa), with a zone of inhibition (ZOI) diameter of 20.6 mm, which was comparable to that of positive gentamicin sulfate with a ZOI value of 25.6 mm. Furthermore, the Day 5 curative activities of both compounds 6 and 6a against kiwifruit bacterial canker were 71.5%, which was higher than those of referred oxine-copper (55.0%) and ethylicin (46.8%) at a concentration of 200 µg mL-1 . In addition, the mechanism analysis based on scanning electron microscopic observation revealed that both compounds 6 and 6a destroyed the integrity of the Psa cell membrane. CONCLUSION: The results of biological tests showed that these bioactive compounds exhibit potent antimicrobial activities, which have the potential to be developed into new antibacterial agents. © 2023 Society of Chemical Industry.

Diversity, antibacterial and phytotoxic activities of culturable endophytic fungi from Pinellia pedatisecta and Pinellia ternata.

BACKGROUND: Endophytic fungi of medicinal plants, as special microorganisms, are important sources of antibacterial compounds. However, the diversity and antibacterial activity of endophytic fungi from Pinellia Tenore have not been systematically studied. RESULTS: A total of 77 fungi were isolated from roots, stems, leaves, and tubers of Pinellia ternata and P. pedatisecta. All fungi were belonged to five classes and twenty-five different genera. Biological activities tests indicated that 21 extracts of endophytic fungi exhibited antibacterial activities against at least one of the tested bacteria, and 22 fermentation broth of endophytic fungi showed strong phytotoxic activity against Echinochloa crusgalli with the inhibition rate of 100%. Furthermore, four compounds, including alternariol monomethyl ether (1), alternariol (2), dehydroaltenusin (3) and altertoxin II (4), and three compounds, including terreic acid (5), terremutin (6), citrinin (7), were isolated from Alternaria angustiovoidea PT09 of P. ternata and Aspergillus floccosus PP39 of P. pedatisecta, respectively. Compound 5 exhibited strong antibacterial activities against Escherichia coli, Micrococcus tetragenus, Staphylococcus aureus, and Pseudomonas syringae pv. actinidiae with the inhibition zone diameter (IZD) of 36.0, 31.0, 33.7, 40.2 mm and minimum inhibitory concentration (MIC) values of 1.56, 3.13, 1.56, 1.56 µg/mL respectively, which were better than or equal to those of positive gentamicin sulfate. The metabolite 7 also exhibited strong antibacterial activity against P. syringae pv. actinidiae with the IZD of 26.0 mm and MIC value of 6.25 µg/mL. In addition, the compound 7 had potent phytotoxic activity against E. crusgalli with the inhibition rate of 73.4% at the concentration of 100 µg/mL. CONCLUSIONS: Hence, this study showed that endophytic fungi of P. ternata and P. pedatisecta held promise for the development of new antibiotic and herbicide resources.

Diversity, antibacterial activity and chemical analyses of gut-associated fungi isolated from the Crocothemis servilia.

Insect-associated fungi are a potentially rich source of novel natural products with antibacterial activity. Here, we investigated the community composition and phylogenetic diversity of gut-associated fungi of the dragonfly (Crocothemis Servilia) using a combination of culture-dependent and culture-independent methods. A total of 42 fungal isolates were obtained from the guts of the dragonfly, which belonged to four classes and thirteen different genera. Amplicon sequencing analyses revealed that the fungal communities were more diverse, and a total of 136 genera were identified and dominated by the genera Wojnowiciella and Phoma. The antibacterial bioassay showed that five fungal crude extracts of representative isolates have shown antibacterial activities. Among them, the extract of Phoma sp. QTH17 showed the best antibacterial activities against Escherichia coli, Micrococcus tetragenus, and Staphylococcus aureus with the disc diameter of inhibition zone diameter (IZD) of 6.50, 10.80, and 8.70 mm, respectively. Chemical analysis of Phoma sp. QTH17 led to the discovery of five known compounds, including ergosterol (1), 3-Chlorogentisyl alcohol (2), epoxydon (3), epoxydon 6-methylsalicylate ester (4) and mannitol (5). Among them, the compound 3 exhibited potent antibacterial activities against E. coli, M. tetragenus, and S. aureus with the IZD of 7.00, 14.00, and 12.50 mm, respectively, which were slightly weaker than those of the positive gentamicin sulfate with the IZD of 11.13, 18.30, and 12.13 mm, respectively. In conclusion, our results confirmed that the diversity of gut-associated fungi of C. Servilia could be expected to explore the resource of new species and antibacterial substances.

Community composition, bacterial symbionts, antibacterial and antioxidant activities of honeybee-associated fungi.

BACKGROUND: Fungi associated with insects represent one potentially rich source for the discovery of novel metabolites. However, a comprehensive understanding of the fungal communities of Apis mellifera ligustica remains elusive. RESULTS: Here, we investigated the phylogenetic diversity and community composition of honeybee-associated fungi using combination of culture-dependent and culture-independent approaches. A total of forty-five fungi were isolated and purified from the Apis mellifera ligustica, royal jelly, and honeycomb, which belonged to four classes and eleven different genera. Furthermore, 28 bacterial 16S rRNA gene sequences were obtained by PCR from the fungal metagenome. High-throughput sequencing analyses revealed that the fungal communities were more diverse, a total of 62 fungal genera were detected in the honeybee gut by culture-independent method, whereas only 4 genera were isolated by culture-dependent method. Similarly, 247 fungal genera were detected in the honeycomb, whereas only 4 genera were isolated. In addition, we assessed the antibacterial and antioxidant activities of fungal isolates. Most fungal crude extracts obtained from the cultivation supernatant exhibited antioxidant activities. Only two fungal crude extracts displayed moderate activity against Escherichia coli and Staphylococcus aureus. Chemical analysis of Chaetomium subaffine MFFC22 led to the discovery of three known compounds, including cochliodinol (1), emodin (2), chrysophanol (3). Among them, cochliodinol (1) showed intense DPPH radical scavenging activity with the 50% inhibitory concentration (IC50) of 3.06 µg/mL, which was comparable to that of the positive ascorbic acid (IC50 = 2.25 µg/mL). Compound 2 displayed weak inhibitory activities against Micrococcus tetragenus and S. aureus. CONCLUSIONS: This research provided a fundamental clue for the complex interactions among honeybees, fungi, bacterial symbionts, and the effects on the honeybee. Furthermore, the diversity of honeybee-associated fungi had great potential in finding the resource of new species and antioxidants.

First Report of Leaf Blight on Mahonia fortunei Caused by Botryosphaeria dothidea in China.

Mahonia fortunei belongs to Berberidaceae, and is often used as a potted plant in Chinese tradition medicine (Li et al. 2015). In March 2019, leaf blight was observed on this species in the campus of Anhui Agricultural University (31°51'51″N; 117°15'31″E) in Hefei City, Anhui Province, China. The average disease incidence was 35% over 20 m2 planting area. Symptoms of the disease are easily apparent, with irregular, dark brown necrosis at the edge of the leaves. To identify the pathogen, symptomatic leaves were collected from three symptomatic leaves from three plants and cut into small pieces. Leaf pieces from the margin of the diseased tissues were surface sterilized for 1 min in 75% ethanol, rinsed three times with sterile distilled water, and subsequently, plated onto potato dextrose agar (PDA) and incubated in dark at 28°C for 4 days. The colonies of three isolates were obtained and appeared morphologically similar in agar media. The cultures were initially white, gradually becoming dark gray, and could grow to the edges of plates (90mm diameter) four days after subculturing. After 7 days on media, the colony was induced for sporulation by UV for 15min, and conidia were observed after 2 weeks. Conidia were nonseptate, hyaline, and oval, measuring 16.9 to 25.3 µm (mean 21.4 µm) × 5.0 to 8.4 µm (mean 6.8 µm) (n = 50). The isolate was identified as Botryosphaeria dothidea preliminarily according to the morphological characteristics (Zhai et al. 2014). For accurate identification, DNA was extracted from the mycelia. The internal transcribed spacer (ITS), ß-tubulin (TUB) and Ef1-a were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b and 983f /2228r, respectively (Zhai et al. 2014). The 543-bp ITS (GenBank accession no. MK850215), 673-bp TUB (no. MN370930) and 1069-bp Ef1-a (no. MN598070) showed >99% identity with those of B. dothidea KP183180 (Xu et al. 2015), KU306116 (Lisboa et al. 2016) and DQ767637.1 (Schoch et al. 2006), respectively. Based on cultural characteristics and phylogenetic analysis, the current isolate associated with leaf blight of M. fortunei was identified as B. dothidea. The representative strain was deposited at the China Center for Type Culture Collection (CCTCC) as CCTCC AF 2019004 SX03. Pathogenicity test was performed on three healthy M. fortunei on campus. Three branches of each plant were selected and all leaves on branches were wounded with dissecting needle by puncturing, then inoculated with a spore suspension (105 conidia/mL) by a hand-held spray bottle, using sterile water as control (Huang et al. 2019). In order to maintain high relative humidity, the inoculated leaves were sealed with transparent plastic bags for 3 days. Fourteen days after inoculation, symptoms (leaf blight) were similar to those on infected leaves on campus, while the controls remained healthy. The experiments were conducted three times and the pathogen was reisolated from inoculated leaves and was confirmed as B. dothidea based on morphological and molecular analyses. To our knowledge, this is the first report of B. dothidea causing marginal leaf necrosis on M. fortunei in China. This study provides a preliminary basis for identifying the causal agent of this plant disease.

Metabolomics-Based Clinical Efficacy of Compound Shenlu Granule, a Chinese Patent Medicine, in the Supportive Management of Aplastic Anemia Patients: A Randomized Controlled Pilot Trial.

OBJECTIVE: To explore the clinical efficacy and mechanism of compound Shenlu granule (SLG) treatment in patients with aplastic anemia (AA). METHODS: A total of 89 AA patients were randomly divided into an SLG supportive group (group A, n = 44) and a control group (group B, n = 45) while continuing Western medical management. After 6 months, hemograms, traditional Chinese medicine (TCM) syndrome scores, and overall clinical efficacy rate were assessed. Serum metabolomics characteristics were observed using ultraperformance liquid chromatography-mass spectrometry after SLG intervention. RESULTS: The levels of red blood cell (RBC), hemoglobin (Hb), and platelet (PLT) were increased in both groups after treatment for 6 months (P < 0.05), and in group A, the elevation of PLT became much more significant (P < 0.01). The TCM syndrome score was lower in group A than in group B after treatment (P < 0.05). Metabolomics data showed a significant difference in the patients using SLG after 6 months, and 14 biomarkers were identified. CONCLUSION: SLG supportive treatment showed positive results in patients with AA, and metabolomics data indicated that SLG influenced aminoacyl-tRNA biosynthesis and glycerophospholipid metabolism to gradually return to normal.

Icariin protects rats against 5/6 nephrectomy-induced chronic kidney failure by increasing the number of renal stem cells.

BACKGROUND: Chronic kidney disease poses a serious health problem worldwide with increasing prevalence and lack of effective treatment. This study aimed to investigate the mechanism of icariin in alleviating chronic renal failure induced by 5/6 nephrectomy in rats. METHODS: The chronic renal failure model was established by a two-phased 5/6 nephrectomy procedure. The model rats were given daily doses of water or icariin for 8 weeks. The kidney morphology was checked by HE staining. The levels of blood urea nitrogen, serum creatinine, and serum uric acid were measured by colometric methods. The expression of specified genes was analyzed by quantitative real-time PCR and immunohistochemical staining. The number of renal stem/progenitor cells was analyzed by CD133 and CD24 immunohistochemical staining. RESULTS: Icariin protected against CDK-caused damages to kidney histology and improved renal function, significantly reduced levels of BUN, creatinine, and uric acid. Icariin inhibited the expression level of TGF-ß1 whereas upregulated HGF, BMP-7, WT-1, and Pax2 expression. Moreover, ccariin significantly increased the expression of CD24, CD133, Osr1, and Nanog in remnant kidney and the numbers of CD133(+)/CD24(+) renal stem/progenitor cells. CONCLUSIONS: These data demonstrated that icariin effectively alleviated 5/6 nephrectomy induced chronic renal failure through increasing renal stem/progenitor cells.