Purification and characterization of crude fructooligosaccharides extracted from red onion (Allium cepa var. viviparum) by yeast treatment.

BACKGROUND: Yeast treatment has been used for purification of fructooligosaccharides (FOSs). However, the main drawback of this approach is that yeast can only partially remove sucrose from crude FOSs. The main objective of this research was to screen yeast strains for the capability of selectively consuming unwanted sugars, namely fructose, glucose, and sucrose, in crude FOSs extracted from red onion (Allium cepa var. viviparum) with minimal effect on FOS content. RESULTS: Among 43 yeast species isolated from Miang, ethnic fermented tea leaves, and Assam tea flowers, Candida orthopsilosis FLA44.2 and Priceomyces melissophilus FLA44.8 exhibited the greatest potential to specifically consume these unwanted sugars. In a shake flask, direct cultivation of C. orthopsilosis FLA44.2 was achieved in the original crude FOSs containing an initial FOSs concentration of 88.3 ± 1.2 g/L and 52.9 ± 1.2 g/L of the total contents of fructose, glucose, and sucrose. This was successful with 93.7% purity and 97.8% recovery after 24 h of cultivation. On the other hand, P. melissophilus FLA48 was limited by initial carbohydrate concentration of crude FOSs in terms of growth and sugar utilization. However, it could directly purify two-fold diluted crude FOSs to 95.2% purity with 92.2% recovery after 72 h of cultivation. Purification of crude FOSs in 1-L fermenter gave similar results to the samples purified in a shake flask. Extracellular ß-fructosidase was assumed to play a key role in the effective removal of sucrose. Both Candida orthopsilosis FLA44.2 and P. melissophilus FLA44.8 showed γ-hemolytic activity, while their culture broth had no cytotoxic effect on viability of small intestinal epithelial cells, preliminarily indicating their safety for food processing. The culture broth obtained from yeast treatment was passed through an activated charcoal column for decolorization and deodorization. After being freeze dried, the final purified FOSs appeared as a white granular powder similar to refined sugar and was odorless since the main sulfur-containing volatile compounds, including dimethyl disulfide and dipropyl trisulfide, were almost completely removed. CONCLUSION: The present purification process is considered simple and straight forward, and provides new and beneficial insight into utilization of alternative yeast species for purification of FOSs.

First Report of Rose Apple Leaf Spot Caused by Colletotrichum siamense in Thailand.

The rose apple (Syzygium samarangense (Blume) Merr. & L.M.Perry) plant has been commonly cultivated in Thailand. In May of 2022, leaf spot disease of rose apple was discovered in Chiang Mai Province, Thailand, with approximately 30% disease incidence. The typical symptoms initially showed brown spots (0.1 to 0.5 mm in diameter) with a yellow halo surrounding. These spots then expanded with black edges and the infected leaves appear blighted and desiccated. In humid conditions, pale yellow conidiomata formed on the lesions. Small pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface disinfected with 1% NaClO for 1 min, 70% ethanol for 30 s, and washed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25 ºC for three days. Three fungal isolates (SDBR-CMU419, SDBR-CMU420, and SDBR-CMU421) were obtained that exhibited similar morphology. Fungal colonies appeared white to gray with cottony mycelia after incubation on PDA at 25 ºC for one week. All fungal isolates produced asexual morph on PDA. Setae were 5590 × 2.53.5 µm, brown with 13-septa, cylindrical base, and tip rounded. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 2050 µm long (n = 50). Conidia were one-celled, hyaline, smooth-walled, aseptate, straight, cylindrical, end round, guttulate, 1017 × 35 µm (n = 50). Appressoria were mostly formed from mycelia, oval to irregular, brown to dark brown, smooth-walled, 610 × 57 µm (n = 50). Morphologically, all fungal isolates resembled to Colletotrichum (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), ß-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. The ITS (ON740892 to ON740894), act (ON759242 to ON759244), tub2 (ON759245 to ON759247), CAL (ON759248 to ON759250), and GAPDH (ON759251 to ON759253) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum phylogenetic analyses indicated that all fungal isolates clustered with C. siamense ICMP 18578 (type strain) with strong statistical (99% ML) support. For pathogenicity test, asymptomatic leaves, stems and fruits detached from healthy plants were surface disinfected using 0.1% NaClO for 3 min, washed three times with sterile distilled water, and air-dried. A uniform wound (3 pores, 1 mm in width) was made at the equator of each leaf, stem and fruit using aseptic needles. Mycelial plugs (5 mm in diameter) and conidia suspensions (1 × 106 conidia/ml) of each fungal isolate grown on PDA at 25 ºC for one week were used to inoculate both wounded and unwounded samples by the detached method (Huda­Shakirah et al. 2022; Suwannarach et al. 2022). Plugs of PDA and sterile distilled water were used as controls. Ten replications were performed for each treatment and the experiment was repeated twice. All inoculated samples were incubated in a moist chamber at 25 ºC with 90% relative humidity. The disease severity index was used to evaluate the specimens (Acar et al. 2008; Ngegba et al. 2017). After one week, both wounded and unwounded leaves that inoculated with mycelial plugs and conidia suspensions showed brown leaf spots and a weak infection. Mycelial plugs inoculated on both wounded and unwounded fruits revealed a moderate infection, but inoculation of conidia suspensions showed a weak infection. No symptoms of disease were observed on the inoculated stems. Control leaves, stems and fruits remained asymptomatic. The pathogen C. siamense was re-isolated from spot and rot lesions on PDA in order to fulfill Koch's postulates. Phoulivong et al. (2012) reported that C. siamense is a causal agent of fruit rot in rose apples cultivated in Lao and Thailand. To our knowledge, this is the first report of C. siamense causing leaf spots on rose apple plants in Thailand. Importantly, these findings will provide crucial information for epidemiologic studies and in the development of appropriate management strategies for this newly emerging disease.

Fungal diversity notes 1387-1511: taxonomic and phylogenetic contributions on genera and species of fungal taxa.

This article is the 13th contribution in the Fungal Diversity Notes series, wherein 125 taxa from four phyla, ten classes, 31 orders, 69 families, 92 genera and three genera incertae sedis are treated, demonstrating worldwide and geographic distribution. Fungal taxa described and illustrated in the present study include three new genera, 69 new species, one new combination, one reference specimen and 51 new records on new hosts and new geographical distributions. Three new genera, Cylindrotorula (Torulaceae), Scolecoleotia (Leotiales genus incertae sedis) and Xenovaginatispora (Lindomycetaceae) are introduced based on distinct phylogenetic lineages and unique morphologies. Newly described species are Aspergillus lannaensis, Cercophora dulciaquae, Cladophialophora aquatica, Coprinellus punjabensis, Cortinarius alutarius, C. mammillatus, C. quercoflocculosus, Coryneum fagi, Cruentomycena uttarakhandina, Cryptocoryneum rosae, Cyathus uniperidiolus, Cylindrotorula indica, Diaporthe chamaeropicola, Didymella azollae, Diplodia alanphillipsii, Dothiora coronicola, Efibula rodriguezarmasiae, Erysiphe salicicola, Fusarium queenslandicum, Geastrum gorgonicum, G. hansagiense, Helicosporium sexualis, Helminthosporium chiangraiensis, Hongkongmyces kokensis, Hydrophilomyces hydraenae, Hygrocybe boertmannii, Hyphoderma australosetigerum, Hyphodontia yunnanensis, Khaleijomyces umikazeana, Laboulbenia divisa, Laboulbenia triarthronis, Laccaria populina, Lactarius pallidozonarius, Lepidosphaeria strobelii, Longipedicellata megafusiformis, Lophiotrema lincangensis, Marasmius benghalensis, M. jinfoshanensis, M. subtropicus, Mariannaea camelliae, Melanographium smilaxii, Microbotryum polycnemoides, Mimeomyces digitatus, Minutisphaera thailandensis, Mortierella solitaria, Mucor harpali, Nigrograna jinghongensis, Odontia huanrenensis, O. parvispina, Paraconiothyrium ajrekarii, Parafuscosporella niloticus, Phaeocytostroma yomensis, Phaeoisaria synnematicus, Phanerochaete hainanensis, Pleopunctum thailandicum, Pleurotheciella dimorphospora, Pseudochaetosphaeronema chiangraiense, Pseudodactylaria albicolonia, Rhexoacrodictys nigrospora, Russula paravioleipes, Scolecoleotia eriocamporesi, Seriascoma honghense, Synandromyces makranczyi, Thyridaria aureobrunnea, Torula lancangjiangensis, Tubeufia longihelicospora, Wicklowia fusiformispora, Xenovaginatispora phichaiensis and Xylaria apiospora. One new combination, Pseudobactrodesmium stilboideus is proposed. A reference specimen of Comoclathris permunda is designated. New host or distribution records are provided for Acrocalymma fici, Aliquandostipite khaoyaiensis, Camarosporidiella laburni, Canalisporium caribense, Chaetoscutula juniperi, Chlorophyllum demangei, C. globosum, C. hortense, Cladophialophora abundans, Dendryphion hydei, Diaporthe foeniculina, D. pseudophoenicicola, D. pyracanthae, Dictyosporium pandanicola, Dyfrolomyces distoseptatus, Ernakulamia tanakae, Eutypa flavovirens, E. lata, Favolus septatus, Fusarium atrovinosum, F. clavum, Helicosporium luteosporum, Hermatomyces nabanheensis, Hermatomyces sphaericoides, Longipedicellata aquatica, Lophiostoma caudata, L. clematidis-vitalbae, Lophiotrema hydei, L. neoarundinaria, Marasmiellus palmivorus, Megacapitula villosa, Micropsalliota globocystis, M. gracilis, Montagnula thailandica, Neohelicosporium irregulare, N. parisporum, Paradictyoarthrinium diffractum, Phaeoisaria aquatica, Poaceascoma taiwanense, Saproamanita manicata, Spegazzinia camelliae, Submersispora variabilis, Thyronectria caudata, T. mackenziei, Tubeufia chiangmaiensis, T. roseohelicospora, Vaginatispora nypae, Wicklowia submersa, Xanthagaricus necopinatus and Xylaria haemorrhoidalis. The data presented herein are based on morphological examination of fresh specimens, coupled with analysis of phylogenetic sequence data to better integrate taxa into appropriate taxonomic ranks and infer their evolutionary relationships.

Co-production of gallic acid and a novel cell-associated tannase by a pigment-producing yeast, Sporidiobolus ruineniae A45.2.

BACKGROUND: Gallic acid has received a significant amount of interest for its biological properties. Thus, there have been recent attempts to apply this substance in various industries and in particular the feed industry. As opposed to yeasts, fungi and bacteria and their tannases have been well documented for their potential bioconversion and specifically for the biotransformation of tannic acid to gallic acid. In this research, Sporidiobolus ruineniae A45.2 is introduced as a newly pigment-producing and tannase-producing yeast that has gained great interest for its use as an additive in animal feed. However, there is a lack of information on the efficacy of gallic acid production from tannic acid and the relevant tannase properties. The objective of this research study is to optimize the medium composition and conditions for the co-production of gallic acid from tannic acid and tannase with a focus on developing an integrated production strategy for its application as a feed additive. RESULTS: Tannase produced by S. ruineniae A45.2 has been classified as a cell-associated tannase (CAT). Co-production of gallic acid obtained from tannic acid and CAT by S. ruineniae A45.2 was optimized using response surface methodology and then validated with the synthesis of 11.2 g/L gallic acid from 12.3 g/L tannic acid and the production of 31.1 mU/mL CAT after 48 h of cultivation in a 1-L stirred tank fermenter. Tannase was isolated from the cell wall, purified and characterized in comparison with its native form (CAT). The purified enzyme (PT) revealed the same range of pH and temperature optima (pH 7) as CAT but was distinctively less stable. Specifically, CAT was stable at up to 70 °C for 60 min, and active under its optimal conditions (40 °C) at up to 8 runs. CONCLUSION: Co-production of gallic acid and CAT is considered an integrated and green production strategy. S. ruineniae biomass could be promoted as an alternative source of carotenoids and tannase. Thus, the biomass, in combination with gallic acid that was formed in the fermentation medium, could be directly used as a feed additive. On the other hand, gallic acid could be isolated and purified for food and pharmaceutical applications. This paper is the first of its kind to report that the CAT obtained from yeast can be resistant to high temperatures of up to 70 °C.

Natural Bioactive Compounds from Fungi as Potential Candidates for Protease Inhibitors and Immunomodulators to Apply for Coronaviruses.

The inhibition of viral protease is an important target in antiviral drug discovery and development. To date, protease inhibitor drugs, especially HIV-1 protease inhibitors, have been available for human clinical use in the treatment of coronaviruses. However, these drugs can have adverse side effects and they can become ineffective due to eventual drug resistance. Thus, the search for natural bioactive compounds that were obtained from bio-resources that exert inhibitory capabilities against HIV-1 protease activity is of great interest. Fungi are a source of natural bioactive compounds that offer therapeutic potential in the prevention of viral diseases and for the improvement of human immunomodulation. Here, we made a brief review of the current findings on fungi as producers of protease inhibitors and studies on the relevant candidate fungal bioactive compounds that can offer immunomodulatory activities as potential therapeutic agents of coronaviruses in the future.

Cultivation of Mushrooms and Their Lignocellulolytic Enzyme Production Through the Utilization of Agro-Industrial Waste.

A large amount of agro-industrial waste is produced worldwide in various agricultural sectors and by different food industries. The disposal and burning of this waste have created major global environmental problems. Agro-industrial waste mainly consists of cellulose, hemicellulose and lignin, all of which are collectively defined as lignocellulosic materials. This waste can serve as a suitable substrate in the solid-state fermentation process involving mushrooms. Mushrooms degrade lignocellulosic substrates through lignocellulosic enzyme production and utilize the degraded products to produce their fruiting bodies. Therefore, mushroom cultivation can be considered a prominent biotechnological process for the reduction and valorization of agro-industrial waste. Such waste is generated as a result of the eco-friendly conversion of low-value by-products into new resources that can be used to produce value-added products. Here, we have produced a brief review of the current findings through an overview of recently published literature. This overview has focused on the use of agro-industrial waste as a growth substrate for mushroom cultivation and lignocellulolytic enzyme production.

Herbicide-tolerant endophytic bacteria of rice plants as the biopriming agents for fertility recovery and disease suppression of unhealthy rice seeds.

BACKGROUND: Dirty panicle disease (DPD) caused by several fungal phytopathogens results in damage and depreciation of rice seeds. Unhealthy rice seeds with DPD are potent reservoirs of pathogens and unable to be used as seed stock as they can spread the disease in the paddy fields leading to the severe loss of rice yield and quality. In this study, we aim to search for beneficial endophytes of commercially cultivated rice plants and utilize them as biostimulants in seed biopriming for fertility recovery and disease suppression of unhealthy rice seeds. RESULTS: Forty-three bacterial endophytes were isolated from rice plants grown in the herbicide-treated paddy fields. Five isolates of these endophytes belonging to the genus Bacillus show excellent antifungal activity against fungal pathogens of DPD. Based on germination tests, biopriming unhealthy rice seeds by soaking in bacterial suspensions for 9 or 12 h was optimal as evidenced by the lowest disease incidence and longer shoot and root lengths of seedlings germinated, compared with controls made of non-treated or hydroprimed healthy and unhealthy seeds. Pot experiments were carried out to evaluate the impact of seed biopriming, in which the percentage of healthy rice yield produced by rice plants emerging from bioprimed seeds was not significantly different, compared to the controls originating respectively from non-treated healthy seeds and chemical fungicide-treated unhealthy seeds. CONCLUSION: Biopriming of unhealthy rice seeds with herbicide-tolerant endophytic bacteria could recover seed fertility and protect the full life cycle of emerging rice plants from fungal pests. With our findings, seed biopriming is a straightforward approach that farmers can apply to recover unhealthy rice seed stock, which enables them to reduce the cost and use of agrochemicals in the commercial production of rice and to promote green technology in sustainable agriculture.

Amycolatopsis eburnea sp. nov., an actinomycete associated with arbuscular mycorrhizal fungal spores.

A novel actinomycete, designated strain GLM-1T, was isolated from arbuscular mycorrhizal fungal spores from Funneliformis mosseae RYA08, collected from Aquilaria crassna Pierre ex Lec. rhizosphere soil in Klaeng, Rayong Province, Thailand. Morphological characteristics of this strain included long chains of rod-like cells and squarish elements. The cell-wall composition of this novel isolate contained meso-diaminopimelic acid. The whole-cell diagnostic sugars were arabinose and galactose. The predominant menaquinone was MK-9(H4). The major fatty acids were iso-C16 : 0 and iso-C15 : 0. Only phosphatidylethanolamine was detected as a polar lipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain GLM-1T was closely related to Amycolatopsis rhabdoformis SB026T (99.11 %) with a low DNA-DNA hybridization value of 22.6-34.7 %. Genome sequencing revealed a genome size of 10 Mbp. There were obvious distinctions in the average nucleotide identity values between stain GLM-1T and its closely related strains at around 86-93 % (ANIb) and 89-94 % (ANIm). The digital DNA-DNA hybridization values between strain GLM-1T and type strains of phylogenetically related species were 34-55 %. The G+C content of the genomic DNA was 71.8 mol%. Based on these data, strain GLM-1T is considered to represent a novel species of the genus Amycolatopsis, for which the name Amycolatopsiseburnea sp. nov. is proposed. The type strain is GLM-1T (=TBRC 9315T=NBRC 113658T).

Optimization and characterization of red pigment production from an endophytic fungus, Nigrospora aurantiaca CMU-ZY2045, and its potential source of natural dye for use in textile dyeing.

Some of the most important natural pigments have been produced from fungi and used for coloring in food, cosmetics, textiles, and pharmaceutical products. Forty-seven isolates of endophytic fungi were isolated from Cinnamomum zeylanicum in northern Thailand. Only one isolate, CMU-ZY2045, produced an extracellularly red pigment. This isolate was identified as Nigrospora aurantiaca based on morphological characteristics and the molecular phylogenetic analysis of a combined four loci (large subunit and internal transcribed spacer of ribosomal DNA, ß-tubulin, and translation elongation factor 1-alpha genes). The optimum conditions for red pigment production from this fungus were investigated. The results indicated that the highest red pigment yield was observed in the liquid medium containing glucose as a carbon source and yeast extract as a nitrogen source, at a pH value of 5.0 and at 27 °C with shaking for 5 days. The crude red pigment revealed the highest level of solubility in methanol. A fungal red pigment was found to have high stability at temperatures ranging from 20 to 50 °C and pH values at a range of 5.0-6.0. Based on liquid chromatography-mass spectrometry analyses, the red pigment was characterized as bostrycin. The extracted pigment was used for the textile dyeing process. Crude fungal red pigment revealed the highest staining ability in cotton fabrics and displayed excellent fastness to washing, which showing negative cytotoxicity at the concentrations used to cell culture. This is the first report on bostrycin production from N. aurantiaca.

Characterization and Dye Decolorization Potential of Two Laccases from the Marine-Derived Fungus Pestalotiopsis sp.

: Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest Km and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms.

Streptomyces venetus sp. nov., an actinomycete with a blue aerial mycelium.

A Gram-positive bacterium, designated CMU-AB225T, was isolated from rhizosphere soil of an oil palm (Elaeis guineensis). The strain exhibited a blue aerial spore mass and a light cream to moderate yellow substrate mycelium and formed chains of spiny spores. Whole-cell hydrolysates consisted of ll-diaminopimelic acid, glucose, ribose, mannose and galactose. The predominant menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The polar lipids profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside, four unidentified lipids, two unidentified aminolipids and an unidentified glycolipid. The major cellular fatty acids (>10 %) were iso-C16 : 0, C16 : 0, anteiso-C15 : 0 and iso-C15 : 0. The G+C content of genomic DNA was 69.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CMU-AB225T was a member of the genus Streptomyces and formed a distinct phyletic line which was most closely related to Streptomyces koyangensis JCM 14915T, Streptomyces misionensis JCM 4497T and Streptomyces aurantiogriseus JCM 4346T. Multilocus sequence analysis (MLSA) using five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) showed that the MLSA distances of strain CMU-AB225T to phylogenetically related species were greater than the 0.007 threshold. Moreover, the low values of DNA-DNA relatedness and phenotypic differences, especially a blue aerial mycelium, enabled strain CMU-AB225T to be distinguished from its closely related species. It is thus proposed that strain CMU-AB225T represents a novel species, namely Streptomyces venetus sp. nov. The type strain is CMU-AB225T (=JCM 31290T=TBRC 2001T).

Amycolatopsis oliviviridis sp. nov., a novel polylactic acid-bioplastic-degrading actinomycete isolated from paddy soil.

A novel bioplastic-degrading actinomycete, strain SCM_MK2-4T, was isolated from paddy soil in Thailand. The 16S rRNA gene sequence showed that strain SCM_MK2-4T belonged to the genus Amycolatopsis, with the highest sequence similarity to Amycolatopsisazurea JCM 3275T (99.4 %), and was phylogenetically clustered with this strain along with Amycolatopsislurida JCM 3141T (99.3 %), A. japonica DSM 44213T (99.2 %), A. decaplanina DSM 44594T (99.0 %), A. roodepoortensis M29T (98.9 %), A. keratiniphilasubsp. nogabecina DSM 44586T (98.8 %), A. keratiniphilasubsp. keratiniphila DSM 44409T (98.5 %), A. orientalis DSM 40040T (98.4 %) and A. regifaucium GY080T (98.3 %). A combination of DNA-DNA hybridization results ranging from 42.8±3.2 to 66.2±1.4 % with the type strains of A. azurea and A. lurida and some different phenotypic characteristics indicated that the strain could be distinguished from its closest phylogenetic neighbours. Whole-cell hydrolysates of the strain were shown to contain meso-diaminopimelic acid, arabinose, galactose, glucose, ribose, mannose, rhamnose and xylose. The predominant menaquinone was MK-9(H4). The major cellular fatty acid profile consisted of iso-C15 : 0, iso-C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2OH) and C16 : 0. The polar lipid composition of the strain consisted of phosphatidyl-N-methylethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylglycerol, aminophospholipids, an unidentified phospholipid and two unidentified glycolipids. The G+C content of the genomic DNA was 68.2 mol%. On the basis of phylogenetic analyses, DNA-DNA hybridization experimentation and the phenotypic characteristics, it was concluded that strain SCM_MK2-4T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis oliviviridis sp. nov. is proposed. The type strain is SCM_MK2-4T (=TBRC 7186T=JCM 32134T).

Pseudonocardia thailandensis sp. nov., an actinomycete isolated from a subterranean termite nest.

A novel Gram-stain-positive bacterium designated CMU-NKS-70T was isolated from a subterranean termite nest and characterized using a polyphasic approach. The strain exhibited branching, pinkish-cream aerial mycelium and cream-brown substrate mycelium, and formed chains of rod-like spores. The 16S rRNA gene sequence analyses indicated that strain CMU-NKS-70T belonged to the genus Pseudonocardia, showing high similarity with Pseudonocardia oroxyli D10T (98.9 % 16S rRNA gene sequence similarity), Pseudonocardia xishanensis YIM 63638T (98.9 %) and Pseudonocardia kujensis A4038T (98.5 %). However, DNA-DNA relatedness values between strains CMU-NKS-70T and the closest phylogenetically related species ranged from 40.5±2.9 to 48.6±0.7 %. Whole-cell hydrolysates of strain CMU-NKS-70T consisted of meso-diaminopimelic acid, glucose, galactose, arabinose, mannose, ribose and rhamnose. The predominant menaquinone was MK-8(H4). The major cellular fatty acids (>10 %) were iso-C16 : 0, C16 : 0, C16 : 1ω7c and/or iso-C15 : 0 2-OH and 10-methyl C16 : 0. The polar lipids detected were phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, three unidentified glycolipids and two unidentified phospholipids. The G+C content of genomic DNA was 71.9 mol%. The physiological and biochemical properties also supported the phenotypic distinction of strain CMU-NKS-70T from its closely related species. On the basis of evidence from this study using a polyphasic approach, strain CMU-NKS-70T represents a novel species of the genus Pseudonocardia for which the name Pseudonocardia thailandensis sp. nov. is proposed. The type strain is CMU-NKS-70T (=JCM 31292T=TBRC 2000T).

Effective enhancement of polylactic acid-degrading enzyme production by Amycolatopsis sp. strain SCM_MK2-4 using statistical and one-factor-at-a-time approaches.

This study aims to find the optimal medium and conditions for polylactic acid (PLA)-degrading enzyme production by Amycolatopsis sp. SCM_MK2-4. Screening of the most effective components in the enzyme production medium by Plackett-Burman design revealed that the silk cocoon and PLA film were the most significant variables enhancing the PLA-degrading enzyme production. After an response surface methodology, a maximum amount of PLA-degrading enzyme activity at 0.74 U mL-1 was predicted and successfully validated at 95% after 0.39% (w/v) silk cocoon and 1.62% (w/v) PLA film were applied to the basal medium. The optimal initial pH value, temperature, and inoculum size were evaluated by a method considering one-factor-at-a-time. The values were recorded at an initial pH in the range of 7.5-9.0, a temperature of 30-32°C, and an inoculum size of 4-10%. The highest activity of approximately 0.95 U mL-1 was achieved after 4 days of cultivation using the optimized medium and under optimized conditions in a shake flask. Upscaling to the use of a 3-L stirred tank fermenter was found to be successful with a PLA-degrading activity of 5.53 U mL-1; which represents a 51-fold increase in the activity compared with that obtained from the nonoptimized medium and conditions in the shake flask.

Daldionin, an Unprecedented Binaphthyl Derivative, and Diverse Polyketide Congeners from a Fungal Orchid Endophyte.

Thailand possesses a rich diversity of orchid species that, in turn, live in symbiosis with a wide variety of fungi. Such endophytes have the potential to produce secondary metabolites with bioactivity against orchid and/or human pathogens. The orchid-associated fungal strain Daldinia eschscholtzii was found to produce a diverse range of aromatic polyketides including the new naphthalene derivatives daldionin, nodulones B and C, and daldinones F and G along with eight known compounds. Daldionin possesses an unprecedented oxane-linked binaphthyl ring system. These compounds demonstrate the high diversity of structural variations that are constructed during fungal biosynthesis, and the results include important observations concerning the biosynthesis of binaphthyl derivatives. Daldionin was found to have weak antiproliferative activity against HUVEC and K-562 cell lines. All but one of the isolated compounds showed moderate antimicrobial activity towards at least one of the four tested microbial strains.

Streptomyces palmae sp. nov., isolated from oil palm (Elaeis guineensis) rhizosphere soil.

Actinomycete strain CMU-AB204T was isolated from oil palm rhizosphere soil collected in Chiang Mai University (Chiang Mai, Thailand). Based on morphological and chemotaxonomic characteristics, the organism was considered to belong to the genus Streptomyces. Whole cell-wall hydrolysates consisted of ll-diaminopimelic acid, glucose, ribose and galactose. The predominant menaquinones were MK-9(H4), MK-9(H6), MK-9(H2) and MK-8(H4). The fatty acid profile contained iso-C15 : 0, iso-C16 : 0 and anteiso-C15 : 0 as major components. The principal phospholipids detected were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C content of strain CMU-AB204T was 70.9 mol%. Based on 16S rRNA gene sequence similarity, strain CMU-AB204T was closely related to Streptomyces orinoci JCM 4546T (98.7 %), Streptomyces lilacinus NBRC 12884T (98.5 %), Streptomyces abikoensis CGMCC 4.1662T (98.5 %), Streptomyces griseocarneus JCM 4905T (98.4 %) and Streptomyces xinghaiensis JCM 16958T (98.3 %). Phylogenetic trees revealed that the new strain had a distinct taxonomic position from closely related type strains of the genus Streptomyces. Spiny to hairy spores clearly differentiated strain CMU-AB204T from the five most closely related Streptomyces species, which produced smooth spores. On the basis of evidence from this polyphasic study, it is proposed that strain CMU-AB204T represents a novel species of the genus Streptomyces, namely Streptomyces palmae sp. nov. The type strain is CMU-AB204T (=JCM 31289T=TBRC 1999T).

The ectomycorrhizal status of a tropical black bolete, Phlebopus portentosus, assessed using mycorrhizal synthesis and isotopic analysis.

Phlebopus portentosus is one of the most popular wild edible mushrooms in Thailand and can produce sporocarps in the culture without a host plant. However, it is still unclear whether Phlebopus portentosus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus. In this study, Phlebopus portentosus sporocarps were collected from northern Thailand and identified based on morphological and molecular characteristics. We combined mycorrhizal synthesis and stable isotopic analysis to investigate the trophic status of this fungus. In a greenhouse experiment, ECM-like structures were observed in Pinus kesiya at 1 year after inoculation with fungal mycelium, and the association of Phlebopus portentosus and other plant species showed superficial growth over the root surface. Fungus-colonized root tips were described morphologically and colonization confirmed by molecular methods. In stable isotope measurements, the δ(13)C and δ(15)N of natural samples of Phlebopus portentosus differed from saprotrophic fungi. Based on the isotopic patterns of Phlebopus portentosus and its ability to form ECM-like structures in greenhouse experiments, we conclude that Phlebopus portentosus could be an ECM fungus.

Evaluation of Muscodor suthepensis strain CMU-Cib462 as a postharvest biofumigant for tangerine fruit rot caused by Penicillium digitatum.

BACKGROUND: This study investigated both the in vitro and in vivo biofumigant ability of the endophytic fungus Muscodor suthepensis CMU-Cib462 to control Penicillium digitatum, the main cause of tangerine fruit rot. RESULTS: Volatile compounds from M. suthepensis inhibited mycelial growth of the pathogen. The most abundant compound was 2-methylpropanoic acid, followed by 3-methylbutan-1-ol. They showed median effective doses (ED50) on P. digitatum growth of 74.91 ± 0.73 and 250.29 ± 0.29 µL L(-1) airspace respectively. Rye grain was found to be a suitable solid medium for M. suthepensis inoculum production. The results indicated that mycofumigation with a 30 g rye grain culture of M. suthepensis for 12 h controlled tangerine fruit rot. The percentage weight loss and soluble solids concentration of fumigated tangerines were similar to those of non-infected and non-fumigated fruits. CONCLUSION: Muscodor suthepensis has potential as a biofumigant for controlling postharvest disease of tangerine fruit.

Monascus purpureus-fermented Thai glutinous rice reduces blood and hepatic cholesterol and hepatic steatosis concentrations in diet-induced hypercholesterolemic rats.

BACKGROUND: Red yeast rice (RYR) is a fermented product used as a food supplement to promote blood circulation and lower blood cholesterol levels in eastern Asia. Interestingly, monacolin K is the most active compound in RYR that proved to inhibit HMG-CoA reductase in the cholesterol biosynthesis pathway. METHODS: The hypocholesterolemic effects of oral administration of Thai RYR, produced by fermentation of Thai glutinous rice (Oryza sativa L. var. Niaw San-pah-tawng) with Monascus purpureus CMU 002U, were determined in normal and hypercholesterolemic rats. The rats were divided into six groups, and fed two different kinds of diet. Groups I-II, normal rats fed with a normal diet (SP-diet), were treated with distilled water (SP-control) and 2.0 g/kg/day of RYR extract (SP-2 g). In Groups III-VI, the rats were rendered hypercholesterolemic by feeding them a high fat and cholesterol diet (HFC-diet), and were treated with distilled water (HFC-control), 1.0 g/kg/day (HFC-1 g), 2.0 g/kg/day (HFC-2 g) of RYR extract, and 5.0 mg/kg/day of rosuvastatin (HFC-rosuvastatin) for 30 days, respectively. RESULTS: The RYR extract significantly decreased the concentrations of serum total cholesterol and low density lipoprotein cholesterol (LDL-C), atherosclerotic index, LDL-C/HDL-C ratio and hepatic cholesterol levels in both HFC-1 g and HFC-2 g groups (p < 0.05) as compared with the HFC-control group, and with no significant change in high density lipoprotein cholesterol (HDL-C) concentrations among all six groups. The reduction of serum TC and LDL-C also paralleled the observed changes in mRNA expressions of the genes involved in cholesterol biosynthesis and homeostasis in the liver. The hypercholesterolemic rats treated with RYR extract were significantly higher in LDLR and HMGR expression, but lower in CYP7A1 expression when compared to the untreated hypercholesterolemic rats (HFC-control) (p < 0.05). The hepatic injuries in hypercholesterolemic rats were also obviously alleviated by RYR extract. CONCLUSIONS: The extract of Thai RYR possessed potent hypocholesterolemic and anti-atherogenic activities in diet-induced hypercholesterolemic rats. The possible mechanism involving cholesterol-lowering potential of the extract might contribute to its ability to increase LDL-C endocytosis in hepatocyte and to competitively inhibit HMG-CoA reductase, a key enzyme for cholesterol biosynthesis in liver.

Isolation and screening of biopolymer-degrading microorganisms from northern Thailand.

Forty agricultural soils were collected from Chiang Mai and Lampang provinces in northern Thailand. Bacteria, actinomycetes and fungi were isolated and screened for their ability to degrade polylactic acid (PLA), polycaprolactone (PCL) and poly(butylene succinate) (PBS) by the agar diffusion method. Sixty-seven actinomycetes, seven bacteria and five fungal isolates were obtained. The majority of actinomycetes were Streptomyces based on morphological characteristic, chemotaxonomy and 16S rRNA gene data. Seventy-nine microorganisms were isolated from 40 soil samples. Twenty-six isolates showed PLA-degradation (32.9 %), 44 isolates showed PBS-degradation (55.7 %) and 58 isolates showed PCL-degradation (73.4 %). Interestingly, 16 isolates (20.2 %) could degrade all three types of bioplastics used in this study. The Amycolatopsis sp. strain SCM_MK2-4 showed the highest enzyme activity for both PLA and PCL, 0.046 and 0.023 U/mL, respectively. Moreover, this strain produced protease, esterase and lipase on agar plates. Approximately, 36.7 % of the PLA film was degraded by Amycolatopsis sp. SCM_MK2-4 after 7 days of cultivation at 30 °C in culture broth.