Soil salinity exert negative impacts on agricultural production and regarded as a crucial issue in global wetland rice production (Oryza sativa L.). Indigenous salt-tolerant plant growth-promoting rhizobacteria (Bacillus sp.) could be used for improving rice productivity under salinity stress. This study screened potential salt-tolerant plant growth-promoting rhizobacteria (PGPR) collected from coastal salt-affected rice cultivation areas under laboratory and glasshouse conditions. Furthermore, the impacts of these PGPRs were tested on biochemical attributes and nutrient contents in various rice varieties under salt stress. The two most promising PGPR strains, i.e., 'UPMRB9' (Bacillus tequilensis 10b) and 'UPMRE6' (Bacillus aryabhattai B8W22) were selected for glasshouse trial. Results indicated that 'UPMRB9' improved osmoprotectant properties, i.e., proline and total soluble sugar (TSS), antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Moreover, 'UPMRB9' inoculated rice plants accumulated higher amount of nitrogen and calcium in tissues. Therefore, the indigenous salt-tolerant PGPR strain 'UPMRB9' could be used as a potential bio-augmentor for improving biochemical attributes and nutrient uptake in rice plants under salinity stress. This study could serve as a preliminary basis for future large-scale trials under glasshouse and field conditions.
Langkocyclines A1-A3 and B1 and B2, five new angucycline antibiotics produced by Streptomyces sp. Acta 3034, were detected in the course of our HPLC-diode array screening. The producing strain was isolated from the rhizospheric soil of a Clitorea sp. collected from Burau Bay, Langkawi, Malaysia, and was characterized by morphological, physiological and chemotaxonomic features in addition to 16S ribosomal RNA gene sequence information. Strain Acta 3034 is closely related to Streptomyces psammoticus NBRC 13971(T) and Streptomyces lanatus NBRC 12787(T). Langkocyclines consist of an angular tetracyclic benz[a]anthracene skeleton and hydrolyzable O-glycosidic sugar moieties. The yellow-colored A-type langkocyclines differ in their aglycon from the blue-lilac-colored B-type langkocyclines. The A-type langkocycline aglycon is identical to that of aquayamycin and urdamycin A. The chemical structures of the langkocyclines were elucidated by HR-MS, 1D and 2D NMR experiments. They are biologically active against Gram-positive bacteria and exhibit a moderate antiproliferative activity against various human tumor cell lines.
Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
The present study aimed to isolate actinobacteria from soil samples and characterized them using molecular tools and screened their secondary metabolites for antimicrobial activities. Thirty-nine strains from four different location of Barrientos Island, Antarctica using 12 types of isolation media was isolated. The isolates were preceded to screening of secondary metabolites for antimicrobial and antifungal activities. Using high-throughput screening methods, 38% (15/39) of isolates produced bioactive metabolites. Approximately 18% (7/39), 18% (7/39), 10% (4/39) and 2.5% (1/39) of isolates inhibited growth of Candida albicans ATCC 10231(T), Staphylococcus aurues ATCC 51650(T), methicillin-resistant Staphylococcus aurues (MRSA) ATCC BAA-44(T) and Pseudomonas aeruginosa ATCC 10145(T), respectively. Molecular characterization techniques like 16S rRNA analysis, Enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), Random amplified polymorphic DNA (RAPD) and composite analyses were used to characterize the actinobacteria strains. Analysis of 16S rRNA sequences is still one of the most powerful methods to determine higher taxonomic relationships of Actinobacteria. Both RAPD and ERIC-PCR fingerprinting have shown good discriminatory capability but RAPD proved to be better in discriminatory power than ERIC-PCR. Our results demonstrated that composite analysis of both fingerprinting generally increased the discrimination ability and generated best clustering for actinobacteria strains in this study.
The aim of the present study was to examine the prevalence of thermophilic Campylobacter spp. (Campylobacter jejuni and Campylobacter coli) in soil, poultry manure, irrigation water, and freshly harvested vegetables from vegetable farms in Malaysia. C. jejuni was detected in 30.4% and 2.7% of the soil samples, 57.1% and 0% of the manure samples, and 18.8% and 3% of the vegetable samples from farm A and farm B, respectively, when using the MPNPCR method. Campylobacter spp. was not found in any of the irrigation water samples tested. Therefore, the present results indicate that the aged manure used by farm A was more contaminated than the composted manure used by farm B. Mostly, the leafy and root vegetables were contaminated. C. coli was not detected in any of the samples tested in the current study. Both farms tested in this study were found to be contaminated by campylobacters, thereby posing a potential risk for raw vegetable consumption in Malaysia. The present results also provide baseline data on Campylobacter contamination at the farm level.
Environmental contamination by petroleum hydrocarbons, mainly crude oil waste from refineries, is becoming prevalent worldwide. This study investigates the bioremediation of water contaminated with crude oil waste. Bacillus salamalaya 139SI, a bacterium isolated from a private farm soil in the Kuala Selangor in Malaysia, was found to be a potential degrader of crude oil waste. When a microbial population of 108 CFU ml-1 was used, the 139SI strain degraded 79% and 88% of the total petroleum hydrocarbons after 42 days of incubation in mineral salt media containing 2% and 1% of crude oil waste, respectively, under optimum conditions. In the uninoculated medium containing 1% crude oil waste, 6% was degraded. Relative to the control, the degradation was significantly greater when a bacteria count of 99 × 108 CFU ml-1 was added to the treatments polluted with 1% oil. Thus, this isolated strain is useful for enhancing the biotreatment of oil in wastewater.
A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
Arsenic is a common contaminant in gold mine soil and tailings. Microbes present an opportunity for bio-treatment of arsenic, since it is a sustainable and cost-effective approach to remove arsenic from water. However, the development of existing bio-treatment approaches depends on isolation of arsenic-resistant microbes from arsenic contaminated samples. Microbial cultures are commonly used in bio-treatment; however, it is not established whether the structure of the cultured isolates resembles the native microbial community from arsenic-contaminated soil. In this milieu, a culture-independent approach using Illumina sequencing technology was used to profile the microbial community in situ. This was coupled with a culture-dependent technique, that is, isolation using two different growth media, to analyse the microbial population in arsenic laden tailing dam sludge based on the culture-independent sequencing approach, 4 phyla and 8 genera were identified in a sample from the arsenic-rich gold mine. Firmicutes (92.23%) was the dominant phylum, followed by Proteobacteria (3.21%), Actinobacteria (2.41%), and Bacteroidetes (1.49%). The identified genera included Staphylococcus (89.8%), Pseudomonas (1.25), Corynebacterium (0.82), Prevotella (0.54%), Megamonas (0.38%) and Sphingomonas (0.36%). The Shannon index value (3.05) and Simpson index value (0.1661) indicated low diversity in arsenic laden tailing. The culture dependent method exposed significant similarities with culture independent methods at the phylum level with Firmicutes, Proteobacteria and Actinobacteria, being common, and Firmicutes was the dominant phylum whereas, at the genus level, only Pseudomonas was presented by both methods. It showed high similarities between culture independent and dependent methods at the phylum level and large differences at the genus level, highlighting the complementarity between the two methods for identification of the native population bacteria in arsenic-rich mine. As a result, the present study can be a resource on microbes for bio-treatment of arsenic in mining waste.
Black pepper production in Malaysia was restricted by various diseases. Hazardous chemical products appear to be the best solution to control diseases in black pepper cultivation. However, persistence of chemical residues in peppercorns could affect the quality of exports and consumptions. Application of fertilizers is crucial to sustain pepper growth and high yield. But, continuous use of chemical fertilizers could affect the soil ecosystem and eventually restrict nutrient uptake by pepper roots. Therefore, we propose biological approaches as an alternative solution instead of chemical products to sustain pepper cultivation in Malaysia. In this study, we have isolated a total of seven indigenous rhizobacteria antagonistic to soil-borne Fusarium solani, the causal fungus of slow decline, the most serious debilitating disease of black pepper in Malaysia. The isolated bacteria were identified as Bacillus subtilis, Bacillus siamensis, Brevibacillus gelatini, Pseudomonas geniculata, Pseudomonas beteli, Burkholderia ubonensis and Burkholderia territorii. These bacteria were effective in production of antifungal siderophore with the amount of 53.4 %-73.5 % per 0.5 mL of cell-free supernatants. The bacteria also produced appreciable amount of chitinase with chitinolytic index was ranged from 1.19 to 1.76. The bacteria have shown phosphate solubilizing index within 1.61 to 2.01. They were also efficient in ACC deaminase (0.52 mM-0.62 mM) and ammonia (60.3 mM-75.3 mM) production. The isolated antagonists were efficacious in stimulation of black pepper plant growth and root development through IAA (10.5 μg/mL-42.6 μg/mL) secretion. In conclusion, the isolated rhizobacteria are potent to be developed not only as biocontrol agents to minimize the utilization of hazardous chemicals in black pepper disease management, but also developed as bio-fertilizers to improve black pepper plant growth due to their capabilities in plant growth-promotion.
Growth and productivity of rice are negatively affected by soil salinity. However, some salt-tolerant rhizosphere-inhabiting bacteria can improve salt resistance of plants, thereby augmenting plant growth and production. Here, we isolated a total of 53 plant-growth-promoting rhizobacteria (PGPR) from saline and non-saline areas in Bangladesh where electrical conductivity was measured as >7.45 and <1.80 dS/m, respectively. Bacteria isolated from saline areas were able to grow in a salt concentration of up to 2.60 mol/L, contrary to the isolates collected from non-saline areas that did not survive beyond 854 mmol/L. Among the salt-tolerant isolates, Bacillus aryabhattai, Achromobacter denitrificans, and Ochrobactrum intermedium, identified by comparing respective sequences of 16S rRNA using the NCBI GenBank, exhibited a higher amount of atmospheric nitrogen fixation, phosphate solubilization, and indoleacetic acid production at 200 mmol/L salt stress. Salt-tolerant isolates exhibited greater resistance to heavy metals and antibiotics, which could be due to the production of an exopolysaccharide layer outside the cell surface. Oryza sativa L. fertilized with B. aryabhattai MS3 and grown under 200 mmol/L salt stress was found to be favoured by enhanced expression of a set of at least four salt-responsive plant genes: BZ8, SOS1, GIG, and NHX1. Fertilization of rice with osmoprotectant-producing PGPR, therefore, could be a climate-change-preparedness strategy for coastal agriculture.
Microbial natural products serve as a good source for antioxidants. The mangrove-derived Streptomyces bacteria have been evidenced to produce antioxidative compounds. This study reports the isolation of Streptomyces sp. MUM273b from mangrove soil that may serve as a promising source of antioxidants and UV-protective agents. Identification and characterization methods determine that strain MUM273b belongs to the genus Streptomyces. The MUM273b extract exhibits antioxidant activities, including DPPH, ABTS, and superoxide radical scavenging activities and also metal-chelating activity. The MUM273b extract was also shown to inhibit the production of malondialdehyde in metal-induced lipid peroxidation. Strong correlation between the antioxidant activities and the total phenolic content of MUM273b extract was shown. In addition, MUM273b extract exhibited cytoprotective effect on the UVB-induced cell death in HaCaT keratinocytes. Gas chromatography-mass spectrometry analysis detected phenolics, pyrrole, pyrazine, ester, and cyclic dipeptides in MUM273b extract. In summary, Streptomyces MUM273b extract portrays an exciting avenue for future antioxidative drugs and cosmeceuticals development.
This review briefly summarizes the geographical distribution and clinical impact of melioidosis, especially in the tropics. Burkholderia pseudomallei (a gram-negative bacterium) is the major causative agent for melioidosis, which is prevalent in Singapore, Malaysia, Thailand, Vietnam, and Northern Australia. Melioidosis patients are increasingly being recognized in other parts of the world. The bacteria are intrinsically resistant to many antimicrobial agents, but prolonged treatment, especially with combinations of antibiotics, may be effective. Despite therapy, the overall case fatality rate of septicemia in melioidosis remains significantly high. Intracellular survival of the bacteria within macrophages may progress to chronic infections, and about 10% of patients suffer relapses. In the coming decades, melioidosis will increasingly afflict travelers throughout many global regions. Clinicians managing travelers returning from the subtropics or tropics with severe pneumonia or septicemia should consider acute melioidosis as a differential diagnosis. Patients with open skin wounds, diabetes, or chronic renal disease are at higher risk for melioidosis and should avoid direct contact with soil and standing water in endemic regions. Furthermore, there are fears that B. pseudomallei may be used as a biological weapon. Technological advancements in molecular diagnostics and antibiotic therapy are improving the disease outcomes in endemic areas throughout Asia. Research and development efforts on vaccine candidates against melioidosis are ongoing.
There are still numerous protein subfamilies within families and superfamilies that do not yet have conclusive empirical experimental evidence providing a specific function. These proteins persist in databases with the annotation of a specific 'putative' function made by association with discernible features in the protein sequence.
Aluminum toxicity is widely considered as the most important limiting factor for plants growing in acid sulfate soils. A study was conducted in laboratory and in field to ameliorate Al toxicity using plant growth promoting bacteria (PGPB), ground magnesium limestone (GML) and ground basalt. Five-day-old rice seedlings were inoculated by Bacillus sp., Stenotrophomonas maltophila, Burkholderia thailandensis and Burkholderia seminalis and grown for 21 days in Hoagland solution (pH 4.0) at various Al concentrations (0, 50 and 100 μM). Toxicity symptoms in root and leaf were studied using scanning electron microscope. In the field, biofertilizer (PGPB), GML and basalt were applied (4 t·ha-1 each). Results showed that Al severely affected the growth of rice. At high concentrations, the root surface was ruptured, leading to cell collapse; however, no damages were observed in the PGPB inoculated seedlings. After 21 days of inoculation, solution pH increased to >6.0, while the control treatment remained same. Field study showed that the highest rice growth and yield were obtained in the bio-fertilizer and GML treatments. This study showed that Al toxicity was reduced by PGPB via production of organic acids that were able to chelate the Al and the production of polysaccharides that increased solution pH. The release of phytohormones further enhanced rice growth that resulted in yield increase.