Biology

A novel Podoviridae lactic acid bacteria (LAB) phage from Nham, a Thai fermented pork sausage, is reported. From a total of 36 samples, 41 isolates of LAB were obtained and employed as hosts for the isolation of phages. From these LAB, only one phage, designated F 22, was isolated. The lactic acid bacterial isolate named N 22, sensitive to phage F 22 infection was identified by an API 50 CHL kit and N 22's complete sequence of the 16S rDNA sequence. BLASTN analysis of the 16S rDNA sequence revealed a 99% similarity to the 16S rDNA sequence of Weissella cibaria in the GenBank database. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of 92 Â 50 nm and 27 nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 HIM)and floating transfers in low calcium (MCF-10F), or by trypsin-Versene ...

Streptomycetes and phages were isolated from soil in different locations of Thailand. The isolated phages, distinguished by host range determination, plaque morphology and phage morphology under transmission electron microscope, were partially purified by single plaque isolation. From host range determination in a variety of phages, phage Roi-1 was selected to serve as an antigen for immunization in rabbits. High titers of phage suspension were prepared by removal of host cells passing through 0.45 µm membrane filter. Five milliliters of the pure phage suspension was injected to three rabbits twice a week for 1 month. Sera from rabbits were subsequently collected and tested for cross reactivation against another 8 phages. The results revealed that phage Nsaw-30 gave a high degree of relatedness to phage Roi-1.

Productions of kanamycin by 3 mutants of Streptomyces kanamyceticus K1 were investigated. The mutant UUNNK1 capable of producing the highest amounts of kanamycin was selected. Optimal conditions for kanamycin production in shaking cultures were found when the UUNNK1 was grown in a KPMB medium consisting of (g/l): starch, 15; soytone, 8; bacto-peptone, 1; MgSO 4 ⋅7H 2 O, 0.5; K 2 HPO 4 , 1; CaCO 3 , 5; NaCl, 3; KCl, 0.5; pH 8.0-8.6, at 30 0 C on a rotary shaker. Under these conditions, within 3 days, the UUNNK1 produced 200 µg/ml of kanamycin which was 14-fold higher than the amount produced by the original strain K1. The production of kanamycin in a 5-litre fermentor was also investigated. Using similar conditions of a shaking flask culture, with agitation speed of 300 rpm, aeration rate at 1.3 vvm, without pH control, UUNNK1 produced the highest amounts, 350 µ g/ml, of kanamycin in 4 days of cultivation. This was 25-fold higher than the yield produced by K1.

Distribution and diversity of actinophages from soil samples collected from several parts of Thailand were investigated. At first, streptomycetes were isolated and used as hosts for isolation of phages. By enrichment procedure, 24 phages were obtained and their hosts were further studied for antimicrobial producing activity. The phages were partially purified by single plaque isolation and then distinguished by plaque morphology and morphology examination under transmission electron microscope. All phages gave differences in size and shape but they all had a hexagonal head and long non-contractile tails. Thus, they were belonged to group B of Bradley's classification and type B1, in Family Siphoviridae of Ackermann and Eisenstark's taxonomy. Determination of host range of phage with 164 streptomycetes strains, it revealed that all phages displayed the different host range pattern by forming plaques on Streptomyces strains and varied from narrow to broad host range. Phage Roi-1 and Yok-15 each gave very broad host range. The phages giving broad host range were brought to further studied. They showed dissimilar patterns in one step growth experiment. Most of them were able to give maximum replication at neutral pH except phage Yok-15, which was at basidic pH. Requirement of cations, Ca 2+ and Mg 2+ , they were also different. Some phages did not require at all, some required small amounts except Yok-15, Sin-27, Nsaw-28 and Nsaw-30 that required high amounts of Ca 2+. Antigenic relationship between the 9 phages and anti-phage Roi-1 serum revealed that Nsaw-30 and Sing-27 were highly inactivated, thus they were closely serological relatedness. SDS-PAGE showed that the phages displayed a different size of protein bands. The number and size of fragments of all phage DNA, which obtained after digestion with restriction enzymes, showed a unique and different molecular weights. The homology of phage DNA was also employed by plaque hybridization. When using DNA of Surat-11, Ray-7, Yok-15 and Roi-1 DNA as probes, it resulted that the homology of phage DNA toward these probes were 5, 4, 4 and 0, respectively. The fate of actinophage Yok-15, and its host bacteria, Streptomyces sp. 15 (isolated from Thai soil as well as phage Yok-15), S. coelicolor, S. griseus and S. viridochromogenes in two kinds of Thai and Japanese sterile soils using batch microcosms, was also investigated. Phage Yok-15 had a very wide host range, and could infect 39 important strains of Streptomyces species from 48 strains tested. In two kinds of Thai soils, phage Yok-15 multiplied in host strain 15 at a maximal titer level, then decreased rapidly in titer level during incubation. However, no multiplication of phage YoK-15 was observed during the incubation with other three streptomycetes in Thai soils, and also with all used streptomycetes in Japanese soil. During incubation with these streptomycetes, the phages decreased in their titer more rapidly than free phages in both Thai and Japanese soils. These results indicated that natures or conditions of soil and strains of host streptomycetes may affect on the survival and multiplication of phages in soil. On the other hand, the survival of host streptomycetes seemed to be strongly affected by the conditions of soil rather than phage infection. Streptomyces sp. 15 was identified by partial 16S rDNA sequences covering the variable region. It resulted that Streptomyces sp. 15 was closely resembled to Streptomyces sp. NRRL 5183 (accession number AJ391815) which has published in year 2000 and has no any report supported since then. Thus, this strain was designated as a new strain (accession number AY128706). To further characterization of phages Yok-15, the phage was sensitive to chloroform, it stabilized at temperature of 4 0 C, at pH 9 for 5 h, and rapidly inactivated by detergents: sodium pyrophosphate and EDTA. In addition, it could multiply in liquid culture with maximum yields at 200 rpm, with initial host and phage concentration at 1.41x 10 8 cfu/ml and 10 6 pfu/ml, respectively, and could survive at pH 5.

Both neem extract and azadirachtin are widely used in agriculture as organic pesticides because they are nontoxic to humans, animals, and the environment. However, their effects on soil microorganisms and plant growth-promoting rhizobacteria (PGPR), which directly affect soil quality, remain largely unexplored. In this study, the effects of neem extract and azadirachtin on the activity of soil microbes and rhizosphere microorganisms was evaluated. We found that 0.1 and 0.4 gmL-1 of the extract and 1.25 and 2.5 µgmL-1 of azadirachtin inhibited the activity of soil microorganisms in vitro. Treating soil with azadirachtin for two months reduced the number of microorganisms present, while two months of treatment with neem extract increased the number of microorganisms in both the soil and the rhizosphere. The phytopathogenic bacterium Pectobacterium carotovorum was more resistant to azadirachtin than Rhizobium sp. Moreover, treatment of mung beans with neem extract or azadirachtin reduced the number of root nodules and Trichoderma asperellum in the rhizosphere, when compared to the control.

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica ‘KDML 105’ was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations o...

Despite a wealth of knowledge on Salmonella phages worldwide, little is known about poultry-associated Salmonella phages from Thailand. Here, we isolated 108 phages from Thai poultry farms that infect Salmonella enterica serovar Typhimurium. Phages STm101 and STm118 were identified as temperate Siphoviridae phages. Genome sequencing and analyses revealed these phages share approximately 96% nucleotide sequence similarity to phage SPN19, a member of the Chi-like virus genus. PCR amplification of the gene encoding capsid protein E of the Chi-like phage was positive for 50% of phage isolates, suggesting a predominance of this phage type among the sampled poultry farms. In addition to the flagella, two phages required the lipopolysaccharide to infect and lyse Salmonella. Furthermore, phylogenomic analysis demonstrated that phages STm101 and STm118 formed a monophyletic clade with phages isolated from Western countries, but not from closer isolated phages from Korea. However, further inv...

A serious problem for Paan-lime production in Thailand is a citrus canker disease caused by the bacterium Xanthomonas citri subsp. citri (Xcc). The use of antagonist against pathogenic Xcc is increasingly becoming popular. In this study, we investigated the effect of pathogenic Xcc and a bacterial antagonist on defense-related gene expression of Paan-lime. The ability of Xcc and the antagonist Pseudomonas aeruginosa SWUC02 to induce defense-related gene expression, including PR-1, Pt14, LRR8, and LOX gene, was assessed. We assured that all four defense-related genes are present in Paan-lime by analyzing the similarity of nucleotide sequences in parts of the genes against other citrus species. Using reverse-transcription PCR (RT-PCR), we showed that the antagonist is able to induce the expression of LRR8 gene at 24 hours post-inoculation, while Xcc induces PR-1 and LOX gene expression at the same time. Our results suggest that these defense-related genes alter their expressions in re...

Taxonomic and systematic studies of bamboos are traditionally based on floral morphology, but this can lead to difficulties in identification because of the irregular reproductive cycle of the bamboos. To overcome such problems several molecular-marker approaches have been used. In this study, eight species of the woody bamboos belonging to the genus Dendrocalamus were employed. For each species, DNA samples of 20 individual plants from different localities were isolated and then pooled to make eight bulks of DNA. Fifty RAPD primers were used to screen all bulked DNA samples. Only five primers yielded consistent and reproducible RAPD band patterns across all 160 individuals. The amplicons were present among five species of Dendrocalamus, but were absent in the other three species. They were cloned, sequenced and subsequently, five pairs of SCAR primers were designed. All SCAR primers were combined in multiplex PCR reactions to unequivocally discriminate five species of Dendrocalamus.

บทคดยอ มนสำปะหลงเปนพชเศรษฐกจทมความสำคญ และมการคดเลอกพนธปลกทมเปอรเซนตแปงสงเพออตสาหกรรม แตพนธปลกทไดรบการพฒนาอาจมลกษณะสณฐานวทยาคลายคลงกน ดงนนการจำแนกพนธปลกจงตองอาศยบคลากรทมความชำนาญ การศกษานมวตถประสงคเพอพฒนาเครองหมายโมเลกลสำหรบระบพนธปลกของมนสำปะหลงไทยจำนวน 16 พนธปลกทไดจากแหลงเชอพนธกรรมในศนยวจยพชไรระยอง โดยนำแถบดเอนเอซงมความแตกตางกนในพนธปลกตางๆ ทไดจากการทำ HAT-RAPD มาโคลนและหาลำดบนวคลโอไทด เพอออกแบบไพรเมอรสำหรบ SCAR ทมความจำเพาะจำนวน 4 ค โดย SCAR marker ทไดสามารถใชจำแนกพนธปลกของมนสำปะหลงได ดงน (1) 308-bp marker และ 850-bp marker ใชระบอตลกษณของพนธปลกระยอง 60 และหานาท ตามลำดบ (2) 414-bp marker ใชระบกลมพนธปลกระยอง 1 ระยอง 11 ระยอง 90 ระยอง 86-13 หวยบง 60 และเกษตรศาสตร 50 (3) 273-bp marker ใชระบกลมพนธปลกระยอง 3 ระยอง 9 และระยอง 72 และ (4) 414-bp marker และ 273-bp marker ใชระบกลมพนธปลกระยอง 5 และหวยบง 80 นอกจากน ยงไดมการพฒนาเทคนค multiplex PCR ทเหมาะสมกบการใชไพรเมอรสำหรบ SCAR พรอมกนทง 4 คในหนงปฏกรยา เพอใหการตรวจสอบดเอนเอเปาหมายมความรวดเรวและประหยดยงขน ABSTRACT Cassava is an important economi...

Distribution and diversity of actinophages from soil samples collected from several parts of Thailand were investigated. At first, streptomycetes were isolated and used as hosts for isolation of phages. By enrichment procedure, 24 phages were obtained and their hosts were further studied for antimicrobial producing activity. The phages were partially purified by single plaque isolation and then distinguished by plaque morphology and morphology examination under transmission electron microscope. All phages gave differences in size and shape but they all had a hexagonal head and long non-contractile tails. Thus, they were belonged to group B of Bradley's classification and type B1, in Family Siphoviridae of Ackermann and Eisenstark's taxonomy. Determination of host range of phage with 164 streptomycetes strains, it revealed that all phages displayed the different host range pattern by forming plaques on Streptomyces strains and varied from narrow to broad host range. Phage Roi-1 and Yok-15 each gave very broad host range. The phages giving broad host range were brought to further studied. They showed dissimilar patterns in one step growth experiment. Most of them were able to give maximum replication at neutral pH except phage Yok-15, which was at basidic pH. Requirement of cations, Ca 2+ and Mg 2+ , they were also different. Some phages did not require at all, some required small amounts except Yok-15, Sin-27, Nsaw-28 and Nsaw-30 that required high amounts of Ca 2+. Antigenic relationship between the 9 phages and anti-phage Roi-1 serum revealed that Nsaw-30 and Sing-27 were highly inactivated, thus they were closely serological relatedness. SDS-PAGE showed that the phages displayed a different size of protein bands. The number and size of fragments of all phage DNA, which obtained after digestion with restriction enzymes, showed a unique and different molecular weights. The homology of phage DNA was also employed by plaque hybridization. When using DNA of Surat-11, Ray-7, Yok-15 and Roi-1 DNA as probes, it resulted that the homology of phage DNA toward these probes were 5, 4, 4 and 0, respectively. The fate of actinophage Yok-15, and its host bacteria, Streptomyces sp. 15 (isolated from Thai soil as well as phage Yok-15), S. coelicolor, S. griseus and S. viridochromogenes in two kinds of Thai and Japanese sterile soils using batch microcosms, was also investigated. Phage Yok-15 had a very wide host range, and could infect 39 important strains of Streptomyces species from 48 strains tested. In two kinds of Thai soils, phage Yok-15 multiplied in host strain 15 at a maximal titer level, then decreased rapidly in titer level during incubation. However, no multiplication of phage YoK-15 was observed during the incubation with other three streptomycetes in Thai soils, and also with all used streptomycetes in Japanese soil. During incubation with these streptomycetes, the phages decreased in their titer more rapidly than free phages in both Thai and Japanese soils. These results indicated that natures or conditions of soil and strains of host streptomycetes may affect on the survival and multiplication of phages in soil. On the other hand, the survival of host streptomycetes seemed to be strongly affected by the conditions of soil rather than phage infection. Streptomyces sp. 15 was identified by partial 16S rDNA sequences covering the variable region. It resulted that Streptomyces sp. 15 was closely resembled to Streptomyces sp. NRRL 5183 (accession number AJ391815) which has published in year 2000 and has no any report supported since then. Thus, this strain was designated as a new strain (accession number AY128706). To further characterization of phages Yok-15, the phage was sensitive to chloroform, it stabilized at temperature of 4 0 C, at pH 9 for 5 h, and rapidly inactivated by detergents: sodium pyrophosphate and EDTA. In addition, it could multiply in liquid culture with maximum yields at 200 rpm, with initial host and phage concentration at 1.41x 10 8 cfu/ml and 10 6 pfu/ml, respectively, and could survive at pH 5.

The Saen Saeb canal, a transportation route in the central of Bangkok, nowadays is polluted with domestic wastewater. People along the canal still use the Saen Saeb water for watering plants. However, the effect of Saen Saeb water usage on plant growth and soil microorganisms is not well understood. The present study showed the effect of Saen Saeb water on the growth of Chinese kale (Brassica alboglabra), tomato (Lycopersicon esculentum), and Dendrobium orchid (Dendrobium Sonia 'Earsakul') at greenhouse and sterile conditions. We found that the Saen Saeb water reduced the growth of Chinese kale and Dendrobium orchid in the greenhouse condition as well as the growth of the Chinese kale and tomato in the sterile condition. Moreover, the Saen Saeb water reduced the amount of rhizosphere microorganisms of the Chinese kale to five times less than that of tap water. Thus, this may affect the plant growth.

Foodborne illnesses caused by wild mushroom poisoning occur globally and have led to food safety concerns. Here, we reported de novo genome assemblies of the six most commonly encountered toxic mushrooms in Thailand. These comprised Amanita brunneitoxicaria, Cantharocybe virosa, Chlorophyllum molybdites, Entoloma mastoideum, Pseudosperma sp. and Russula subnigricans. The nuclear genome sizes of these species ranged from 40 to 77 Mb, with the number of predicted genes ranging from 5,375 to 14,099. The mitogenome sizes varied from 41,555 to 78,907 bp. The resulting draft genomes of these poisonous mushrooms provide insights into toxin-related genes that may be used to establish genetic markers for monitoring mushroom poisoning outbreaks.

Lactic acid bacteria (LAB) from Thai fermented meat and fish products were isolated. From a total of 93 samples, 152 isolates of lactic acid bacteria were obtained. Antimicrobial activity screening was performed using the agar spot test and the agar well diffusion method. Of the six isolates which produced antimicrobial activities against Weissella confusa N31, only isolate N23 was identified as Weissella cibaria (GenBank accession number AB494716.1) with 99% similarity by 16S rDNA sequence analyses. Complete inactivation of antimicrobial activity produced by W. cibaria N23 was observed after treatment of the bacteriocins with trypsin, actinase, protease XIII, ficin, trypsin from porcine pancreas, a-chymotrypsin and pepsin. In addition, the inhibitory activities were not affected by the addition of catalase. Taken together, these results confirmed that the inhibitory compounds produced by this strain were proteinaceous in nature and possessed typical characteristics of bacteriocins. The highest yield of bacteriocin produced by W. cibaria N23 was recorded at 20 h. The bacteriocin N23 remained stable after 2 h of incubation at pH values between 2.0 and 8.0, and also for 15 min at 121 C. The bacteriocin produced by W. cibaria N23 was found to have a narrow antibacterial spectrum, being able to inhibit only W. confusa N31. In addition, bacteriocin N23 did not adhere to the surface of the producer cells. The results produced from this study will contribute to the existing body of knowledge and enhance the databases of bacteriocin-producing Weissella.

Both neem extract and azadirachtin are widely used in agriculture as organic pesticides because they are nontoxic to humans, animals, and the environment. However, their effects on soil microorganisms and plant growth-promoting rhizobacteria (PGPR), which directly affect soil quality, remain largely unexplored. In this study, the effects of neem extract and azadirachtin on the activity of soil microbes and rhizosphere microorganisms was evaluated. We found that 0.1 and 0.4 gmL-1 of the extract and 1.25 and 2.5 µgmL-1 of azadirachtin inhibited the activity of soil microorganisms in vitro. Treating soil with azadirachtin for two months reduced the number of microorganisms present, while two months of treatment with neem extract increased the number of microorganisms in both the soil and the rhizosphere. The phytopathogenic bacterium Pectobacterium carotovorum was more resistant to azadirachtin than Rhizobium sp. Moreover, treatment of mung beans with neem extract or azadirachtin reduced the number of root nodules and Trichoderma asperellum in the rhizosphere, when compared to the control.

Extensive surveys of mangrove foliose lichens in Caliciaceae and Physciaceae on the Gulf of Thailand revealed eight species of the genera Dirinaria, Physcia, and Pyxine. Species density was highest in the mid-intertidal zone (46%), followed by the landward and seaward zones (31% and 23%, respectively). Fifty-one new internal transcribed spacer sequences were generated and the resulting phylogenies based on maximum likelihood and Bayesian approaches yielded monophyletic lineages of all three genera. However, within the Dirinaria clade formation of polyphyletic assemblages among D. aegialita, D. applanata, and D. picta indicated the presence of homoplasies in certain morphological traits used to characterize them. To address species boundaries of these lichens in Dirinaria picta species complex, methods of DNA barcode-based delineation of putative species were employed. Additional sampling of the Dirinaria species from elsewhere is required to provide further insight into species delimitation of this heterogeneous genus.

บทคัดย่อ   เห็ดปะการังสกุล Ramaria จำนวน 10 ตัวอย่าง จัดจำแนกชนิดตามลักษณะทางสัณฐานวิทยาได้ 9 สปีชีส์ ดังนี้ Ramaria aff. vinosimaculans (PKWS15-74 และ PKWS15-109), R. flava var. aurea (PKWS15-173), Ramaria sp.1 (PKWS15-92), Ramaria sp.2 (PKWS15-95), Ramaria sp.3 (PKWS15-164), Ramaria sp.4 (PKWS15-181), Ramaria sp.5 (PKWS15-194), Ramaria sp.6 (PKWS15-221) และ Ramaria sp.7 (PKWS14-02) เมื่อนำมาสกัดด้วยสารละลายเอทานอลและทดสอบคุณสมบัติทางชีวภาพพบว่าสารสกัดจากเห็ดทุกตัวอย่างมีประสิทธิภาพในการต้านอนุมูลอิสระที่ดีโดยเฉพาะ PKWS15-164 ซึ่งมีค่าใกล้เคียงกับ positive control นอกจากนี้ยังตรวจพบสารฟลาโวนอยด์และสารประกอบฟีนอลในปริมาณที่สูง เช่น PKWS15-164 (3,909.52±58.56 g QE/g extract) และ PKWS15-194 (3,765.88±33.75 g QE/g extract) ซึ่งสอดคล้องกับประสิทธิภาพในการต้านอนุมูลอิสระ ทั้งนี้เนื่องจากสารฟลาโวนอย์เป็นสารต้านอนุมูลอิสระที่ดี ส่วนประสิทธิภาพในการยับยั้งการเจริญของแบคทีเรียพบว่าสารสกัดส่วนใหญ่สามารถยับยั้งการเจริญของ Staphylococcus aureus ได้มากที่สุดรองลงมาคือ Escherichia coli, Bacillus ...

ABSTRACTAeromonas dhakensisis the most virulentAeromonasspecies in tropical and subtropical areas and causes a variety of human diseases. Owing to its resistance against some antibiotics, there is an urgent need for new strategies against this pathogen. This is the first study to isolate and characterize lytic phages againstA. dhakensis. OfAeromonasisolates, only isolate AM could be used to isolate phages using the enrichment technique. This strain was identified via biochemical tests, 16S rDNA sequencing, and whole-genome analyses. All results confirmed that strain AM wasA. dhakensis. In silicodetection of antimicrobial resistance genes and virulence factors corresponding to the main bacterial virulence determinants were observed inA. dhakensisAM, which was then used as the host for phage isolation. Four lytic phages, designated vB_AdhS_TS3, vB_AdhM_TS9, vB_AdhM_DL, and vB_AdhS_M4, were isolated. Transmission electron micrographs showed that vB_AdhS_TS3 and vB_AdhS_M4 belonged toSi...

A recent discovery of Amanita brunneitoxicaria revealed that this mushroom and A. exitialis are the main causative agents of fatal mushroom poisoning in Thailand. Poisonous amatoxins present are bicyclic octapeptides encoded by the MSDIN family and macrocyclized by prolyl oligopeptidase B enzyme. Diversity of the MSDIN toxin-associated genes in A. brunneitoxicaria however, remains unexplored. Therefore, this study aimed to characterize the MSDIN family members in A. brunneitoxicaria in comparison with A. exitialis based on whole genome sequencing, followed by gene annotation using bioinformatic tools. Identification of conserved core peptides of the MSDIN family revealed the presence of α-amanitin and β-amanitin as endogenous toxins in A. brunneitoxicaria. In addition to the major amatoxins, amanexitide and phallacidin were also found in A. exitialis. Phylogenetic analyses of the MSDIN family members showed that an unknown peptide present in A. brunneitoxicaria is clustered with pha...