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.

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.

Stability evaluation of gardenia yellow pigment in the presence of different antioxidants or microencapsulating agents.

The chemical instability of gardenia yellow pigment (GYP) limits its utilization in the food industry. In this study, the effects of different antioxidants (0.2% of tea polyphenols, sodium phytate, potassium citrate, and ascorbic acid) and microencapsulating agents (gum Arabic, maltodextrin, inulin, and gum Arabic/maltodextrin) on the degradation of GYP under different conditions (heat, light, and ferric iron) were evaluated. Then, the characteristic properties of microcapsules coated with gum Arabic/maltodextrin, gum Arabic/maltodextrin/tea polyphenols, maltodextrin, and maltodextrin/tea polyphenols were investigated. Furthermore, food models were simulated to evaluate the GYP stability of the microcapsules. The results showed that tea polyphenols, maltodextrin, and gum Arabic/maltodextrin significantly improved the GYP stability. Moreover, the presence of GYP in microcapsules was confirmed by nuclear magnetic resonance and Fourier transform infrared spectroscopy. In addition, GYP-MD/TP possessed high thermal stability under different cooking methods. PRACTICAL APPLICATION: Gardenia yellow pigment (GYP) is easily degraded under light and high-temperature conditions, which limits its applications in the food industry. This study will provide effective clues for expanding the practical applications of GYP in the natural pigment industry.

Termite Nest Associated Bacillus siamensis YC-9 Mediated Biocontrol of Fusarium oxysporum f. sp. cucumerinum.

The antagonistic potential of bacteria obtained from the nest of Odontotermes formosanus was assessed against Fusarium oxysporum f. sp. cucumerinum (FOC). Of 30, seven termite nest-associated bacteria strains had biocontrol potential. Among them, the strain YC-9 showed the strongest antifungal activity toward FOC. Phylogenetic analysis of the 16S rRNA amplified product of YC-9 revealed its identification as Bacillus siamensis. The in vivo antifungal activity experiment showed that the application of YC-9 at 108 cfu/ml significantly reduced the cucumber wilt incidence with a control efficacy of 73.2%. Furthermore, plant growth parameters such as fresh weight, dry weight, plant height, and root height were significantly improved by 42.6, 53.0, 20.8, and 19.3%, respectively. We found that inoculation with B. siamensis YC-9 significantly increased the activity of defensive enzymes such as peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) in diseased cucumber roots, thereby raising the resistance. PCR using gene-specific primers revealed that B. siamensis YC-9 contains biosynthetic genes for known antibiotics, including bacillomycin, iturin, and surfactin. Chemical analysis of the cultivation of B. siamensis YC-9 resulted in the isolation of five metabolites, including hexadecanoic acid (1), cyclo-(L-phenylalanylglycine) (2), cyclo-(L-trans-Hyp-L-Leu) (3), C15-surfactin (4), and macrolactin A (5), the structures of which were identified by the analysis of NMR spectroscopic data and MS. Among them, the compound 4 showed significant antifungal activity against conidial germination of FOC with an IC50 value of 5.1 µg/ml, which was comparable to that of the positive control, cycloheximide (IC50 value of 2.6 µg/ml). Based on these findings, this study suggests that termite-nest associated B. siamensis YC-9 could be a potential biological control agent for integrated control of soil-borne diseases like cucumber Fusarium wilt.

Pigments of aminophenoxazinones and viridomycins produced by termite-associated Streptomyces tanashiensis BYF-112.

Termite-associated Streptomyces tanashiensis BYF-112 was found as a potential source for yellow and green pigments, which were stable under the tested temperature, light and metal ions. Eight metabolites (1-8), including four new natural yellow pigments aminophenoxazinones (1-4), and two rarely iron dependent green pigments viridomycin A and F (9-10) were isolated from BYF-112 cultured in YMS and YMS treated with FeSO4, respectively. The metabolites 2-4 displayed a significant safety performance on the normal liver cell line L-02, while the metabolite 1 showed weak cytotoxicity against the L-02 and several cancer cells. Especially, in the filter paper disc tests, the compound 1 possessed strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with the zone of inhibition (ZOI) of 15.3 mm, which was equal to that of referenced levofloxacin (ZOI = 15.2 mm). And the metabolite 1 also showed moderate antibacterial activities against Micrococcus teragenus and S. aureus, with the ZOI values of 15.3 and 17.2 mm. In addition, by the minimum inhibitory concentration (MIC) assay, the compound 1 displayed potential antibacterial activities against M. teragenus, S. aureus and MRSA, with the MIC values of 12.5, 12.5, and 25.0 µg/ml, respectively. The present results indicate that BYF-112 may be a promising source for safe and bioactive pigments, which can be used for further development and industrial applications.

Stability evaluation of gardenia yellow pigment in presence of different phenolic compounds.

Gardenia yellow pigment (GYP) may undergo chemical degradation under different conditions resulting in color fading. This study investigated the effects of different phenolic compounds (caffeic acid, rosmarinic acid, tannic acid, epicatechin, chlorogenic acid, epigallocatechin, and epigallocatechin gallate) on the physical and chemical stability of GYP under light and different temperatures. Furthermore, food models with GYP/phenolic compounds were simulated to evaluate the GYP stability under different cooking methods. The addition of phenolic compounds, especially tannic acid, epigallocatechin gallate, epigallocatechin, and rosmarinic acid, significantly improved the GYP stability during light and thermal treatments. Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy confirmed that the formation of hydrogen bonds between GYP and selected phenolic compounds (tannic acid, epigallocatechin gallate, epigallocatechin, and rosmarinic acid), which may lead to the enhancement of GYP stability. Moreover, these selected phenolic compounds provided potent protective effects on GYP under different cooking methods.

Antibacterial Potential of Termite-Associated Streptomyces spp.

Twenty-one strains of termite-associated actinomycetes were tested for their activities against three bacteria. The results showed that nine strains showed bacteriostatic activities against at least one tested bacterium, and the actinomycete YH01, which was isolated from the body surface of the queen of Odontotermes formosanus, had potent antibacterial activity. The YH01 was further identified as Streptomyces davaonensis. Two metabolites roseoflavin (1) and 8-methylamino-8-demethyl-d-riboflavin (2) were isolated and purified from S. davaonensis YH01. Their structures were determined by NMR, MS, and the related literature. The metabolite 1 showed strong inhibition activities against Bacillus subtilis (MIC = 1.56 µg/mL) and Staphylococcus aureus (MIC = 3.125 µg/mL), which were comparable to referenced gentamycin sulfate, with MIC values of 1.56 and 1.56 µg/mL, respectively. Furthermore, the anti-MRSA potential of compound 1 was determined against nine kinds of MRSA strains, with inhibition zones in the ranges of 12.7-19.7 mm under a concentration of 15 µg/6 mm discs and 18.3-22.7 mm under a concentration of 30 µg/6 mm discs. However, metabolite 1 had no inhibitory effect on Gram-negative bacteria. These results suggested that roseoflavin produced by YH01 holds promise for use against Gram-positive bacteria, especially to MRSA.

Chemical compositions and antimicrobial activities of the essential oil from Pterocarya stenoptera C. DC.

The present study investigated the chemical compositions and antimicrobial activities of essential oil from Pterocarya stenoptera. The essential oil obtained by hydrodistillation from the dried leaves of P. stenoptera was analysed by GC-MS and 39 constituents accounting for the total 90.44% of the oil were identified. The main constituents were δ-cadinene (24.83%), caryophyllene oxide (9.10%), α-cadinol (7.48%) and ß-elemene (6.24%). The antimicrobial activity of the oil was evaluated by disc diffusion and broth microdilution methods. The essential oil was found to show broad-spectrum antimicrobial activities against all the tested microorganisms. Bacillus subtilis was the most sensitive strain with the minimum inhibitory concentration of 0.23 mg/mL. The results suggested that the essential oil was a potential source of natural antimicrobials in food preservation and pharmaceutical industry.

Multiple heavy metal tolerance and removal by an earthworm gut fungus Trichoderma brevicompactum QYCD-6.

Fungal bioremediation is a promising approach to remove heavy-metal from contaminated water. Present study examined the ability of an earthworm gut fungus Trichoderma brevicompactum QYCD-6 to tolerate and remove both individual and multi-metals. The minimum inhibitory concentration (MIC) of heavy metals [Cu(II), Cr(VI), Cd(II) and Zn(II)] against the fungus was ranged 150-200 mg L-1 on composite medium, and MIC of Pb(II) was the highest with 1600 mg L-1 on potato dextrose (PD) medium. The Pb(II) presented the highest metal removal rate (97.5%) which mostly dependent on bioaccumulation with 80.0%, and synchronized with max biomass (6.13 g L-1) in PD medium. However, on the composite medium, the highest removal rate was observed for Cu(II) (64.5%). Cellular changes in fungus were reflected by TEM analysis. FTIR and solid-state NMR analyses indicated the involvement of different functional groups (amino, carbonyl, hydroxyl, et al.) in metallic biosorption. These results established that the earthworm-associated T. brevicompactum QYCD-6 was a promising fungus for the remediation of heavy-metal wastewater.

Dragonfly-Associated Trichoderma harzianum QTYC77 Is Not Only a Potential Biological Control Agent of Fusarium oxysporum f. sp. cucumerinum But Also a Source of New Antibacterial Agents.

A strain isolated from the gut of Pantala flavescens was characterized as Trichoderma harzianum QTYC77. The strain was assessed as a potential biocontrol agent against Fusarium oxysporum f. sp. cucumerinum (FOC). Mycoparasitism and competing abilities of T. harzianum QTYC77 lead to inhibition of the mycelial growth of FOC, with the inhibition rate of 70.99%, in dual culture assays. Activities of chitinase and ß-1,3-glucanase, responsible for fungal cell-wall degradation, were gradually increased and their activities were the maximum on the fifth day of fermentation with 23.20 and 1.84 U/mL, respectively. T. harzianum QTYC77 was discovered to have potent biocontrol potential with the control efficiency of 67.43% against the FOC in vivo pot experiment. Furthermore, two novel compounds azaphilone D (1) and E (2) along with three known metabolites 3-hydroxymethyl-6, 8-dimethoxycoumarin (3), harzianone (4), and pachybasin (5) were isolated and identified from T. harzianum QTYC77. Unfortunately, these metabolites did not show antifungal activities against FOC. However, both metabolites 1 and 3 displayed moderate activity against Staphylococcus aureus with disc diameters of zone of inhibition (ZOI) of 7.3 and 7.2 mm, respectively, compared with that of referenced gentamycin (ZOI = 14.5 mm). In addition, metabolite 1 possessed a moderate antibacterial activity against Bacillus subtilis with a ZOI value of 7.0 mm compared with that of positive gentamycin (ZOI = 15.2 mm). The present results suggested that T. harzianum QTYC77 was not only a potential biofungicide against FOC but also the source of new antibacterial agents.

Antibacterial and cytotoxic metabolites of termite-associated Streptomyces sp. BYF63.

Four anthraquinone derivatives, termstrin A, B, C and D (1-4), were isolated and purified from termite-associated Streptomyces sp. BYF63. Their structures were elucidated on the basis of extensive spectroscopic analyses (HR-ESI-MS, 1D and 2D NMR). Compounds 1 and 4 were found to possess potent antibacterial activities against Staphylococcus aureus, with the zone of inhibition (ZOI) values of 12.85 and 11.17 mm, respectively, which were comparable to that of penicillin sodium with ZOI of 13.15 mm. Furthermore, metabolite 1 showed moderate cytotoxicities against melanoma cell line A375 and gastric cancer cell line MGC-803, with IC50 values of 22.76 and 36.65 µM, respectively, which were less than those of referenced adriamycin.

Diversity, Bacterial Symbionts, and Antimicrobial Potential of Termite-Associated Fungi.

The phylogenetic diversity of fungi isolated from the Odontotermes formosanus was investigated by dilution-plate method, combined with morphological characteristics and 5.8S rDNA sequencing. Thirty-nine fungi were isolated and purified from O. formosanus, which were belonging to two phyla and four classes (Sordariomycetes, Dothideomycetes, Eurotiomycetes, Agaricomycetes). Furthermore, nine bacterial 16S rRNA sequences were obtained from total fungal genomic DNA. All bacterial symbionts were segmented into four genera: Bacillus, Methylobacterium, Paenibacillus, and Trabulsiella. The antimicrobial activities of all endophytic fungi extracts were tested by using the filter paper method against Escherichia coli (ATCC 8739), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), and Canidia albicans (ATCC 10231). The results exhibited that 25 extracts (64%) exhibited antibacterial activity against at least one of the tested bacterial strains. Furthermore, the secondary metabolites 1 [5-hydroxyramulosin (1a):biatriosporin M (1b) = 2:1] from the Pleosporales sp. BYCDW4 exhibited potent antimicrobial activities against E. coli, C. albicans, B. subtilis, and S. aureus with the inhibition zone diameter (IZD) of 13.67, 14.33, 12.17, and 11.33 mm, respectively, which were comparable with those of the positive control. 1-(2,5-Dihydroxyphenyl)-3-hydroxybutan-1-one (2) from the Microdiplodia sp. BYCDW8 showed medium inhibitory activities against B. subtilis and S. aureus, with the IZD range of 8.32-9.13 mm. In conclusion, the study showed the diversity of insect symbionts could be expected to develop the resource of new species and antibiotics.

Diversity and antagonistic potential of Actinobacteria from the fungus-growing termite Odontotermes formosanus.

43 Actinobacteria were isolated from the nest of Odontotermes formosanus. A phylogenetic analysis of 23 Actinobacteria isolates with different morphotypes showed that they did not form a monophyletic group. Antifungal bioassays exhibited that many strains inhibit both the termite cultivar Termitomyces and the competitor Xylaria. However, Actinobacteria inhibited the competitor Xylaria more severely than the termite cultural fungus Termitomyces. Furthermore, two Actinobacteria (Streptomyces sp. T33 and S. bellus T37) had a selective antifungal effect on Xylaria, with the inhibition zone of 25.5 and 8.9 mm, respectively. An actinomycin D was isolated from the strain T33 and had potent antifungal activity against Xylaria with IC50 value of less than 3.1 µg/mL. In addition, further bioassays showed that actinomycin D possessed potent antifungal activities against Magnaporthe grisea (IC50 = 0.9 µg/mL), Fusarium oxysporum f. sp. cucumerinum (IC50 = 2.2 µg/mL), Valsa mali (IC50 = 1.7 µg/mL), Rhizoctonia solani (IC50 = 10.3 µg/mL), Dothiorella gregaria (IC50 = 12.5 µg/mL) and F. oxysporum f. sp. mornordicae (IC50 = 14.3 µg/mL), which were comparable to those of referenced cycloheximide. The findings of the present study suggest that the termite-associated Actinobacteria have a potential to be used as microbial fungicide.

Antimicrobial Metabolites Produced by Penicillium mallochii CCH01 Isolated From the Gut of Ectropis oblique, Cultivated in the Presence of a Histone Deacetylase Inhibitor.

Three chemical epigenetic modifiers [5-azacytidine, nicotinamide, and suberoylanilide hydroxamic acid (SAHA)] were applied to induce the metabolites of Penicillium mallochii CCH01, a fungus isolated from the gut of Ectropis oblique. Metabolite profiles of P. mallochii CCH01 were obviously changed by SAHA treatment. Four metabolites (1-4), including two new natural sclerotioramine derivatives, isochromophilone XIV (1) and isochromophilone XV (2), and two known compounds, sclerotioramine (3) and (+)-sclerotiorin (4), were isolated and purified from P. mallochii CCH01 treated with SAHA. Their structures were determined by spectroscopic analyzes. Anti-phytopathogenic activities of the isolated compounds 1-4 were investigated under laboratory conditions, and compound 4 showed broad and important inhibition activities against Curvularia lunata (IC50 = 2.1 µg/mL), Curvularia clavata (IC50 = 21.0 µg/mL), Fusarium oxysporum f. sp. Mornordica (IC50 = 40.4 µg/mL), and Botryosphaeria dothidea (IC50 = 27.8 µg/mL), which were comparable to those of referenced cycloheximide, with IC50 values of 0.3, 5.0, 12.4, and 15.3 µg/mL, respectively. Ingredients 2 and 3 showed selective and potent activities against Colletotrichum graminicola with IC50 values of 29.9 and 9.7 µg/mL, respectively. Furthermore, the antibacterial bioassays showed that compounds 3 and 4 exhibited strong inhibition activities against Bacillus subtilis, with disc diameters of zone of inhibition (ZOI) of 9.1 mm for both compounds, which were a bit weaker than that of referenced gentamycin with a ZOI of 10.8 mm. Additionally, the new metabolite 1 showed a promising activity against Candida albicans (ZOI = 10.5 mm), comparable to that of positive amphotericin B with a ZOI of 23.2 mm. The present results suggest that chemical epigenetic modifier induction was a promising approach to obtaining antimicrobial metabolites encoded by silent biosynthetic genes of P. mallochii.

Extraction optimisation, purification and major antioxidant component of red pigments extracted from Camellia japonica.

The optimised extraction conditions of red pigments (RP) from Camellia japonica obtained with an orthogonal design L9(34) were solid/liquid ratio, temperature, pH and extraction time as 1/10, 60°C, 1.5 and 4h, respectively. The RP were then purified by the macroporous resin method, which showed the resin LX-68 was appropriate for purifying the pigments from C. japonica. The antioxidant activities of these pigments were also investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical in vitro model systems. The DPPH scavenging activity of pigment extract was comparable to that of standard butylated hydroxyanisole (BHA). The IC50 values of the RP and BHA were 4.55 and 4.17µgml-1, respectively. The pigments showed higher hydroxyl radical-scavenging activities than that of mannitol at the same concentration. Following activity-oriented separation, (-)-epicatechin was isolated as an active principle, which exhibited excellent DPPH free radical scavenging activities with IC50 5.08µgml-1.

Phytotoxic and antifungal metabolites from Curvularia sp. FH01 isolated from the gut of Atractomorpha sinensis.

Two main phytotoxic and antifungal phthalic acid butyl isobutyl ester (1) and radicinin (2) were isolated from the culture of Curvularia sp. FH01, a fungus residing in the Atractomorpha sinensis gut. The structures of isolated metabolites were established on the basis of spectral analysis. Metabolites 1 and 2 exhibited significant phytotoxic activity against the radical growth of Echinochloa crusgalli with their IC(50) values of 61.9 and 5.9 µg/mL, respectively, which were comparable to that 2,4-dichlorophenoxyacetic acid (2.0 µg/mL) used as a positive control. The antifungal test results showed that compound 2 possessed strong antifungal activity against Magnaporthe grisea (IC(50)=16.3 µg/mL) and Valsa mali (IC(50)=18.2 µg/mL). The findings of the present study suggest that bioactive properties of the fungus FH01 can be attributed to its major components, phthalic acid butyl isobutyl ester and radicinin, and both agents have a potential to be used as herbicide and fungicide.