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.

Identification of Obscurolide-Type Metabolites and Antifungal Metabolites from the Termite-Associated Streptomyces neopeptinius BYF101.

Streptomyces spp. are well-known symbiotic microorganisms that produce antimicrobial metabolites against various pathogens. We isolated actinomycetes from the body surface of the termite Odontotermes formosanus and identified it as Streptomyces neopeptinius BYF101 based on 16S rRNA phylogenetic analysis. Chemical analysis of the cultures of termite-associated S. neopeptinius BYF101 via HR-MS2 and GNPS analyses enabled the isolation and identification of 20 metabolites, including the unreported obscurolide-type metabolites (1-3). The chemical structures of unreported compounds (1-3) were elucidated using HR-ESI-MS and 1D and 2D NMR analysis, and their absolute configurations were determined via chemical reactions followed by the application of competing enantioselective acylation (CEA) and computational methods for ECD and DP4+ probability calculation. The isolated compounds (1-20) were tested to determine their antifungal activity against two human fungal pathogens, Candida albicans and Cryptococcus neoformans. Among the compounds tested, indole-3-carboxylic acid (9) displayed antifungal activity against C. neoformans, with an MIC value of 12 µg/mL.

Diversity and antimicrobial activities of culturable actinomycetes from Odontotermes formosanus (Blattaria: Termitidae).

BACKGROUND: Actinomycetes are important microbes, and they are very important for developing active substances for useful drugs. Actinomycetes are numerous inhabitants, and they are widely distributed in the nest of fungus-growing termites. Previously, we isolated and purified numerous actinomycetes from the combs of Odontotermes formosanus and obtained a variety of valuable natural products. RESULTS: Here, we isolated and purified actinomycetes from fungus-growing termite Odontotermes formosanus using medium-based cultures. Among the eight media tested, M7 and I-HV media were found suitable for isolating actinomycetes. Further, 84 actinomycetes, including 79 Streptomyces isolates, were isolated and purified from O. formosanus and its combs, which belong to four genera (Streptomyces, Kribbella, Amycolatopsis, and Cellulosimicrobium). Then, the type and quantity of actinomycetes were positively correlated with the activity range of termites. Twenty-two actinomycetes strains showed antimicrobial activities. Among them, the BYF18, BYF48, BYF70, and BYF106 strains exhibited antifungal activities against five pathogenic fungi, with zone of inhibition (ZOI) values ranging from 3 to 21 mm. Grincamycin N was isolated and purified from the metabolites of Streptomyces lannensis (BYF106), and it displayed antibacterial activities against Staphylococcus aureus (ZOI = 13.82 ± 0.52 mm) and Micrococcus tetragenus (ZOI = 17.6 ± 0.5 mm) (gentamycin sulfate, as the positive control, had ZOI values of 19.9 ± 0.5 mm and 30.83 ± 0.75 mm, against S. aureus and M. tetragenus, respectively). CONCLUSIONS: Our results confirmed that the actinomycetes associated with O. formosanus are important sources of new active 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 Spot Caused by Alternaria alternata on Ligustrum japonicum in China.

Ligustrum japonicum is a small evergreen tree belonging to the Oleaceae and widely grown in China as a landscape ornamental and medicinal plant (Oh et al. 2021). In April 2021, a leaf spot disease was observed on Ligustrum japonicum in the campus of Anhui Agricultural University (31°51'4″N; 117°14'54″E), in Hefei City, Anhui Province, China. Approximately 50% of L. japonicum infections showed symptoms of round to oval, brown to dark brown lesions surrounded by a yellow halo. Diseased leaves were collected to determine the caused pathogen. Nine tissue pieces from three symptomatic leaves were surface sterilized with 2% NaClO for 1-2 minutes, followed by 75% ethanol for 1 minute, and then rinsed with sterile water for three times. The tissues samples were plated on potato dextrose agar（PDA）medium and incubated at 28 °C for 3 days. Seven fungal isolates were obtained from the plated tissues; the fungal hyphae were initially white and finally gray brown with flocculent aerial mycelia. Conidia were solitary or in chains, with various shapes, mostly subglobose. The size was (12.0-30.0) µm long and (6.0～12.0) µm wide (n=20). The cultural and morphological characteristics of these isolates were similar to those of Alternaria alternata (Simmons et al. 2007; Garibaldi et al. 2020). For accurate identification, genomic DNA was extracted from the mycelia of representative isolate (JSNZ). The internal transcribed spacer (ITS), 18S nrDNA (SSU), translation elongation factor 1-alpha (Tef1-α) and endopolygalacturonase (endoPG) sequences were amplified with the primer pairs of ITS1/ITS4, NS1/NS4, EFI-728F/EFI-986R and EPG-specific/EPG-3b, respectively (Woudenberg et al, 2013). The sequences were deposited in GeneBank under accessions MZ360963 (ITS), MZ677478 (SSU), OK274117(Tef1-α) and OK513186 (endoPG). BLAST analysis of the sequences of ITS, SSU, Tef1-α and endoPG showed >99% identity with those of A. alternata MK108918(561/601bp), KX609765 (1030/1035bp), LC132712 (281/281bp) and MT185591(459/483bp), respectively. A neighbor-joining phylogenetic tree was generated based on the concatenated data from sequences of ITS, SSU and Tef1-α using MEGA5.1, which clustered the present isolate with A. alternata strain CBS916.96 with high bootstrap support (100%). Based on cultural characteristics and phylogenetic analysis, the current isolate associated with leaf spot of L. japonicum was identified as A. alternata. Pathogenicity test was performed on three healthy L. japonicum on campus. Three healthy leaves of each plant were wounded with one sterile needle and inoculated with a 5-mm-diameter mycelial plug using sterile PDA plugs as control. The inoculated plants were covered with plastic bags and sprayed with water every 24 hours to maintain a high temperature and humidity environment. The experiment was repeated three times. After 12-days of incubation, symptoms were apparent on pathogen-inoculated plants, while the control plants remained asymptomatic. A. alternata was reisolated from inoculated leaves and matched the morphological and molecular characteristics of the original isolates, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of a leaf spot disease caused by A. alternata in L. japonicum in China. Its identification will establish a foundation for managing the disease in China.

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.

First Report of Powdery Mildew Caused by Erysiphe berberidis on Berberis fortunei in China.

Berberis fortune (Lindl.) is commonly used in Chinese traditional medicine (Liu et al. 2020). In April 2020, white powdery colonies covering up to 100% of both upper leaf surfaces and calyces were observed on this species growing on Anhui Agricultural University campus (31°51'51″N; 117°15'31″E) in Hefei City, Anhui Province, China. Sporulating mycelia were white and effuse. Conidiophores were erect, with straight, cylindrical foot cells, 20 to 26 × 9 to 12 µm (average: 24 × 11 µm) (n = 30), followed by one to three shorter cells, and producing conidia in chains. Conidia were ellipsoid-ovoid, subcylindrical, and measured 27 to 36 × 12 to 16.5 µm (average: 32.4 × 14.1 µm) (n = 50). For accurate identification, DNA was extracted from the mycelia, which were collected by scraping symptomatic leaves. The internal transcribed spacer (ITS) was amplified and sequenced using primers ITS1/ITS4. The 623-bp ITS (GenBank accession no. MT449013) showed 99% identity with those of Erysiphe berberidis LC010057 (Takamatsu et al. 2015), KY661153 and KY660920. Based on morphological characteristics and phylogenetic analysis, the powdery mildew fungus on B. fortunei was identified as E. berberidis (Glawe, D. A. 2003). Ten leaves on an asymptomatic B. fortunei were inoculated by gently pressing diseased leaves against the surface of healthy leaves. Ten non-inoculated plants served as controls. All plants were maintained in a greenhouse at 22 to 25°C and >80% relative humidity. Inoculated plants developed powdery mildew colonies after 14 days, whereas uninoculated plants remained healthy. Morphological and molecular characters of the powdery mildew fungus on artificially inoculated plants were identical to those on naturally infected B. fortune. Previously in Siberia, Russia, powdery mildew on woody plants has been reported to be caused by E. berberidis (Tomoshevich M. A. 2019). However, this is the first report of powdery mildew caused by E. berberidis on B. fortunei in China. Its identification will establish a foundation for controlling the disease in China.

Phytotoxic and Antifungal Metabolites from Curvularia crepinii QTYC-1 Isolated from the Gut of Pantala flavescens.

Four metabolites (1⁻4), including a new macrolide, O-demethylated-zeaenol (2), and three known compounds, zeaenol (1), adenosine (3), and ergosta-5,7,22-trien-3b-ol (4) were isolated and purified from Curvularia crepinii QTYC-1, a fungus residing in the gut of Pantala flavescens. The structures of isolated compounds were identified on the basis of extensive spectroscopic analysis and by comparison of the corresponding data with those reported in the literature previously. The new compound 2 showed good phytotoxic activity against Echinochloa crusgalli with an IC50 value of less than 5 µg/mL, which was comparable to that of positive 2,4-dichlorophenoxyacetic acid (2,4-D). Compound 1 exhibited moderate herbicidal activity against E. crusgalli with an IC50 value of 28.8 µg/mL. Furthermore, the new metabolite 2 was found to possess moderate antifungal activity against Valsa mali at the concentration of 100 µg/mL, with the inhibition rate of 50%. These results suggest that the new macrolide 2 and the known compound 1 have potential to be used as biocontrol agents in agriculture.

Phytotoxic, Antifungal and Immunosuppressive Metabolites from Aspergillus terreus QT122 Isolated from the Gut of Dragonfly.

Insect gut microbes have been considered as a resource for bioactive metabolites. The aim of this study was to characterize the compounds of a fungus Aspergillus terreus QT122 associated with the gut of dragonfly. Five main phytotoxic, antifungal, and immunosuppressive substances were isolated from the fungus QT122. The structures of such compounds were identified as emodin (1), 1-methyl emodin (2), terrein (3), methyl 6-acetyl-4-methoxy-7,8-dihydroxynaphthalene-2-carboxylate (4), and dihydrogeodin (5) on the basis of spectroscopic analysis and by comparison of the corresponding data to those reported in the literature previously. The compound 3 exhibited the best phytotoxic activity against the radicle growth of A. retroflexus L. and E. crusgalli L. with their IC50 values of 11.2 and 3.1 µg/mL, which were comparable to that of the positive control of 2,4-dichlorophenoxyacetic acid (2,4-D) with the IC50 values of 8.1 and 1.6 µg/mL, respectively. The compounds 2-3 showed potent antifungal activity in the growth of Alternaria solani with the IC50 value of less than 0.1 µg/mL and the compound 2 also had great inhibitory effect against the growth of Fusarium oxysporum f. sp. cucumerinum (IC50 < 0.1 µg/mL), which was comparable to that of referenced cycloheximide with IC50 value of below 0.1 µg/mL. The compounds 3-5 exhibited strong immunosuppressive activities against the T cell viability with the inhibition rates of more than 99%, which were comparable to positive cyclosporin A under the concentration of 20 µM. These results suggest that the compounds 2-5 have the potential to be used as bio-control agents in agriculture or immunosuppressive agents.

[Phytotoxic metabolite from QTYC01, a fungus residing in the gut of Pantala flavescens larvae].

Objective: To isolate the fungus with phytotoxic activity from the gut of Pantala flavescens larvae and find the phytotoxic lead compound from the fungal metabolites. Methods: QTYC01 was isolated from the gut of P. flavescens larvae by means of spread plate and identified by 5.8S rRNA sequence analysis and morphologic observation. Phytotoxic activities of the fermentation broth and ethyl acetate extracts against the radical growth of weeds as well as the safety of crude extract to the selected crops were tested by Petri dish bioassay. The herbicidal activity of QTYC01 against Echinochloa crusgalli seedlings was carried out by potted bioassay. Fermentation product was purified by recrystallization and identified by extensive spectroscopic analysis. Results: QTYC01 was identified as Curvularia crepinii. The fermentation broth of QTYC01 significantly inhibited the radical growth of E. crusgalli and Amaranthus retroflexus with the inhibition rate of 95.0% and 90.1%, respectively. The fermented liquid showed significant inhibitory activity to the seedling of E. crusgalli with the victimization rate of 71.1%. Under the concentration of 100 µg/mL, ethyl acetate extracts exhibited significant phytotoxic activities against the radical growth of E. crusgalli and A. retroflexus with inhibitory rates of 56.8% and 71.2%, respectively, and showed good security to the selected common crops with the inhibition rate of lower than 33%. Therewith, a bioactive compound was isolated from the ethyl acetate extract and determined as (5Z)-7-oxozeaenol. The compound showed good phytotoxic activity against A. retroflexus with the IC50 value of 4.8 µg/mL. Conclusion: Strain QTYC01 could be potentially developed as a new microbial herbicide.

[Identification and phytotoxic activity of fungus QTYC-51 from the gut of Pantala flavescens larvae].

Objective: To isolate the fungus with phytotoxic activity from the gut of Pantala flavescens larvae. Methods: Strain QTYC-51 was identified by morphological observation and 5.8S rDNA-ITS sequence analysis. Petri dish bioassay was used to test the phytotoxic activity of fermentation broth and monomer compounds of strain QTYC-51 on Echinochloa crusgalli and Amaranthus retroflexus. Bioactive components were isolated from ethyl acetate extracts via chromatographic methods, and the structures were determined by mass spectrum and nuclear magnetic resonance analyses. Results: QTYC-51 was identified as Paraconiothyrium sp.. The fermentation broth had good phytotoxic activity on radical growth of E. crusgalli and A. retroflexus with the inhibition rates of 76.9% and 56.5%, respectively. Five monomer compounds were purified from the fermentation products, including 1,8-dihydroxyanthraquinone, 1-hydroxy-10-methoxy-dibenz[b,e]oxepin-6,11-dione, hydroxyvertixanthone, globosuxanthone and 1,3,6,8-tetrahydroxyanthraquinone. At the concentration of 100 µg/mL, compound globosuxanthone was found to possess obvious phytotoxic effects on radical growth of E. crusgalli and A. retroflexus with the inhibition rates of 94.1% and 79.0%, respectively, which were comparable to that of positive control 2,4-dichlorophenoxyacetic acid. Compound 1-hydroxy-10-methoxy-dibenz[b,e] oxepin-6,11-dione showed potent phytotoxic activity against E. crusgalli and A. retroflexus with inhibition rates of 50.3% and 58.6%, respectively. Conclusion: Strain QTYC-51 could be potentially developed as a microbial herbicide.

Synthesis and antifungal activities of novel polyheterocyclic spirooxindole derivatives.

A series of spirooxindole tetrahydrofuran derivatives 3 were obtained in moderate to good yields via oxindole derivatives 1 and ß-arylacrylonitrile derivatives 2via base-mediated cascade [3 + 2] double Michael reactions under mild conditions and the application of this method in the synthesis of bioactive analogues, such as functionalized spirooxindole octahydrofuro[3,4-c]pyridine derivatives 4 which contain two new heterocyclic rings and two quaternary carbon centers, has also been developed. Subsequently, antifungal activities of all of the synthesized compounds were evaluated against five phytopathogenic fungi (Rhizoctonia solani, Fusarium semitectum, Alternaria solani, Valsa mali and Fusarium graminearum) using the mycelium growth rate method. The preliminary results showed that the spirooxindole octahydrofuro[3,4-c]pyridine derivative 4 showed higher growth inhibition of Valsa mali and Fusarium graminearum, than spirooxindole tetrahydrofuran derivatives 3. For example, spirooxindole octahydrofuro[3,4-c]pyridine derivative 4ab, having a bromine atom at the meta position of the benzene ring, was the best compound in inhibiting F. g. with an IC50 value of 3.31, in particular with inhibition of 4ab on F. g. being similar to that of the control cycloheximide (IC50 = 3.3 µg mL(-1)).

[Inhibition of Amaranthus retroflexus by wasp gut fungal isolate Fusarium oxysporum MF06].

OBJECTIVE: To study the inhibitory effect of wasp gut fungus against the radicle growth of Amaranthus retroflexus for the development of herbicides from microorganisms. METHODS: Eleven strains were isolated from wasp gut. Among them the fermentation broth of strain MF06 showed potent herbicidal activity against A. retroflexus. MF06 was identified by morphological observation and molecular biology identification. Fermentation product was isolated and purified by silica column chromatography, TLC and Sephadex LH-20 column chromatography. Metabolite 1 was obtained from fermentation product, and the inhibitory effect of metabolite 1 against the radicle growth of A. retrofexus was studied. The structure was determined by mass spectrum and nuclear magnetic resonance analyses. RESULTS: By the morphological observation and ITS sequence analysis, MF06 was identified as Fusarium oxysporum. The ethyl acetate extract of MF06 had strong activity against A. retrofexus with inhibition rate of more than 68% under the concentration of 100 µg/mL. It is better than other polarities crude extracts. Metabolite 1 was separated from ethyl acetate extract, and it was determined as a mixture of fusaric acid and 9 ,10-dehydrofusaric acid. The mixture is co-crystallizing in a 1:1 molar stoichiometry. It inhibited radical growth of A. retroflexus with IC50 value of (0.51 ± 0.18) µg/mL, comparable to that of 2-( 2,4- dichlorophenoxy) acetic acid (0.30 ± 0.14 µg/mL) used as a positive control. CONCLUSION: Strain MF06 could be potentially developed as a microbial herbicide.

Screening of endophytic fungi with anti-phytopathogen activities from Heptacodium miconioides.

OBJECTIVE: To find anti-phytopathogen compounds from endophytic fungi associated with the endangered species Heptacodium miconioides. METHODS: Fungi from H. miconioides with antifungal activities were isolated according to the plate growth inhibition method. The fungus with preferable antifungal activities was identified by morphological identification and 5. 8S rRNA sequence analysis. The bioactive metabolites were isolated and purified by chromatographic methods; the structures were determined by spectroscopic analysis. RESULTS: Alternaria solani QZH 10 showed better antifungal activity against Rhizoctorzia solani and Valsa mali with the inhibition rates of 89.1% and 67.9%, respectively. The ethyl acetate crude extract of QZH 10 had strong antifungal activity against Magnaporthe oryzae with the rate of 100. 0% under the concentration of 100 µg/mL. Two antifungal metabolites altersolanol A and 6-O-methylalaternin were isolated and determined from QZH 10. Altersolanol A possessed strong activity against M. oryzae with the inhibition rate of more than 85%, 6-O-methylalaternin had the mightily activity against V. mali with the inhibition rate of 100.0% under the concentration of 100 µg/mL. CONCLUSION: Altersolanol A and 6-O-methylalaternin are potential fungicides originated from microorganisms.

[Isolation and identification of termitarium antagonistic actinomycetes BYC 01 and its active metabolites].

OBJECTIVE: We isolated actinomyces from the termitarium and studied its metabolites to find the antimicrobial compounds. METHODS: We determined the taxonomic status of target strain BYC 01 by morphological observation and 16s rRNA sequence analysis. Growth rate method and agar disc diffusion assays were used to test the antimicrobial activities. Fermentation product was isolated and purified by various chromatographic methods, and the structure was determined by mass spectrum and nuclear magnetic resonance analyses. RESULTS: BYC 01 was identified as Streptomyces violaceoruber. The main antimicrobial ingredients of BYC 01 fermentation broth consisted in the ethyl acetate fraction of moderate polar part. The ethyl acetate extract of BYC 01 had strong antifungal activities against Valsa mali with inhibition rate of more than 90%, and activities against Rhizoctonia solani and Dothiorella gregaria with inhibition rate of more than 60% under the concentration of 100 microg/mL. Furthermore, the extract showed the intermediate antimicrobial activities against Candida albicans, Staphyloccocus aureus, Escherichia coli, Bacillus subtilis and Xanthomonas oryzae with the mean halo diameters ranging from 11.3 to 16.5 mm under the concentration of 30 microg/filter paper. A monomer compound was purified from the fermentation products, and was identified as fogacin on the basis of mass spectrum and nuclear magnetic resonance analyses. The compound fogacin and the positive control had similar antimicrobial activities against C. albicans with inhibition zone of 19.3 mm and 20.1 mm under the concentration of 30 microg/filter paper. CONCLUSION: Strain BYC 01 could be potentially developed as a new antimicrobial agent.

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.

Hosts manipulate lifestyle switch and pathogenicity heterogeneity of opportunistic pathogens in the single-cell resolution.

Host-microbe interactions are virtually bidirectional, but how the host affects their microbiome is poorly understood. Here, we report that the host is a critical modulator to regulate the lifestyle switch and pathogenicity heterogeneity of the opportunistic pathogens Serratia marcescens utilizing the Drosophila and bacterium model system. First, we find that Drosophila larvae efficiently outcompete S. marcescens and typically drive a bacterial switch from pathogenicity to commensalism toward the fly. Furthermore, Drosophila larvae reshape the transcriptomic and metabolic profiles of S. marcescens characterized by a lifestyle switch. More importantly, the host alters pathogenicity and heterogeneity of S. marcescens in the single-cell resolution. Finally, we find that larvae-derived AMPs are required to recapitulate the response of S. marcescens to larvae. Altogether, our findings provide an insight into the pivotal roles of the host in harnessing the life history and heterogeneity of symbiotic bacterial cells, advancing knowledge of the reciprocal relationships between the host and pathogen.

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.

Altitude as a key environmental factor shaping microbial communities of tea green leafhoppers (Matsumurasca onukii).

IMPORTANCE: Host-associated microbial communities play an important role in the fitness of insect hosts. However, the factors shaping microbial communities in wild populations, including environmental factors and interactions among microbial species, remain largely unknown. The tea green leafhopper has a wide geographical distribution and is highly adaptable, providing a suitable model for studying the effect of ecological drivers on microbiomes. This is the first large-scale culture-independent study investigating the microbial communities of M. onukii sampled from different locations. Altitude as a key environmental factor may have shaped microbial communities of M. onukii by affecting the relative abundance of endosymbionts, especially Wolbachia. The results of this study, therefore, offer not only an in-depth view of the microbial diversity of this species but also an insight into the influence of environmental factors.