Solid-state cultivation of Bacillus thuringiensis R 176 with shrimp shells and rice straw as a substrate for chitinase production

In this study, shrimp shell powder, prepared by treating shrimp-processing waste by boiling and crushing, was used as a substrate for isolation of chitinase-producing microorganism. These organisms may have an important economic role in the biological control of rice and other fungal pathogens. Two hundred strains of bacteria with the ability to degrade chitin from shrimp shell waste were isolated from paddy soil, and of these, 40 strains showed chitinase activity in a solid state cultivation. One of the most potent isolates (strain R 176) was identified as Bacillus thuringiensis. Identification was carried out using morphological and biochemical properties along with 16S rRNA sequence analysis. This strain was able to produce high levels of extracellular chitinase in solid media containing shrimp shells as sole carbon source [1.36 U/g initial dry substrate (IDS)], which was 0.36-fold higher than the productivity in a liquid culture with colloidal chitin. The effects of medium composition and physical parameters on chitinase production by this organism were studied. The optimal medium contained shrimp shell mixed with rice straw in 1:1 ratio added with ball-milled chitin 0.5 % (w/v) and ammonium sulfate 0.5 % (w/v). The highest enzyme production (3.86 U/g IDS) by B. thuringiensis R 176 was obtained at pH 7, 37 °C after 14 days growth. With respect to the high amount of chitinase production by this strain in a simple medium, this strain could be a suitable candidate for the production of chitinase from chitinous solid substrates, and further investigations into its structure and characteristics are merited.     

Screening and Optimization of Indole-3-Acetic Acid Production and Phosphate Solubilization from Rhizobacteria Aimed at Improving Plant Growth

A total of 216 bacterial strains were isolated from rice rhizospheric soils in Northern Thailand. The bacterial strains were initially tested for solubilization of inorganic phosphate, indole acetic acid (IAA) production, selected strains were then tested for optimized conditions for IAA production and whether these caused stimulatory effects on bean and maize seedling growth. It was found that all strains had solubilized inorganic phosphate (P), but only 18.05% produced IAA. The best IAA producer was identified by biochemical testing and 16S rDNA sequence analysis as Klebsiella SN 1.1. In addition to being the best IAA producer, this strain was a high P-solubilizer and produced the highest amount of IAA (291.97 ± 0.19 ppm) in culture media supplemented with l-tryptophan. The maximum production of IAA was achieved after 9 days of incubation. The culture requirements were optimized for maximum IAA production. The tested of IAA production by selected isolates was studied in a medium with 0, 0.1, 0.2, 0.5, 0.7, and 0.9% (v/v) l-tryptophan. Low levels (12.6 ppm) of IAA production was recorded without tryptophan addition. Production of IAA in Klebsiella SN 1.1 increased with an increase to 0.2% (v/v) tryptophan concentration. The production of IAA was further confirmed by extraction of crude IAA from this isolate and subsequent Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same R _f value. The Klebsiella strain SN 1.1 also demonstrated stimulatory effects on bean seedlings in vivo.     

Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in Thailand

Rice (Oryza sativa) is a staple food in Thailand and, in addition, feeds around one half of the world’s population. Therefore, diseases of rice are of special concern. Rice is destroyed by 2 main pathogens, Fusarium oxysporum and Pyricularia oryzae the causative agents of root rot and blast in rice respectively. These pathogens result in low grain yield in Thailand and other Southeast Asian countries. Soil samples were taken from paddy fields in Northern Thailand and bacteria were isolated using the soil dilution plate method on Nutrient agar. Isolation yielded 216 bacterial isolates which were subsequently tested for their siderophore production and effectiveness in inhibiting mycelial growth in vitro of the rice pathogenic fungi; Alternaria sp., Fusarium oxysporum, Pyricularia oryzae and Sclerotium sp., the causal agent of leaf spot, root rot, blast and stem rot in rice. It was found that 23% of the bacteria isolated produced siderophore on solid plating medium and liquid medium, In dual culture technique, the siderophore producing rhizobacteria showed a strong antagonistic effect against the Alternaria (35.4%), Fusarium oxysporum (37.5%), Pyricularia oryzae (31.2%) and Sclerotium sp. (10.4%) strains tested. Streptomyces sp. strain A 130 and Pseudomonas sp. strain MW 2.6 in particular showed a significant higher antagonistic effect against Alternaria sp. while Ochrobactrum anthropi D 5.2 exhibited a good antagonistic effect against F. oxysporum. Bacillus firmus D 4.1 inhibited P. oryzae and Kocuria rhizophila 4(2.1.1) strongly inhibited Sclerotium sp. P. aureofaciens AR 1 was the best siderophore producer overall and secreted hydroxamate type siderophore. This strain exhibits an in vitro antagonistic effect against Alternaria sp., F. oxysporum and P. oryzae. Siderophore production in this isolate was maximal after 15 days and at an optimal temperature of 30°C, yielding 99.96 ± 0.46 μg ml^?1 of siderophore. The most effective isolates were identified by biochemical tests and molecular techniques as members of the Genus Bacillus, Pseudomonas and Kocuria including B. firmus D 4.1, P. aureofaciens AR1 and Kocuria rhizophila 4(2.1.1). The study demonstrated antagonistic activity towards the target pathogens discussed and are thus potential agents for biocontrol of soil borne diseases of rice in Thailand and other countries.     

Prevalence of Helicobacter in Laboratory Micein Thailand

Prevalence of Helicobacter is mostly unknown in laboratory animals in Thailand. The 221 mice feces/cecum from 8 universities, 2 pharmaceutical companies and 3 research institutions in Thailand were surveyed for the prevalence and distribution of Helicobacter species by using the Electrochemical DNA chip. Helicobacter were detected 23/46 samples in Specific Pathogen Free (SPF) and 168/175 in conventional condition. Prevalence of Helicobacter were 98%, 96%, 92% and 78% in South (n=40), Northeast (n=40), North (n=25) and Central area (n=116), respectively. Only Central area holds SPF facility resulting in Helicobacter prevalence that seems to be lower than other areas. Three species of Helicobacter were detected in feces/cecum samples by sequence analysis: H. rodentium (67.0%, 148 samples), Helicobacter sp. MIT 01-6451 (15.4%, 34 samples), and unidentified Helicobacter species (14.1%, 9 samples). The results suggested that H. rodentium is the most common species of Helicobacter in laboratory mice in Thailand.     

Phosphate solubilization potential and stress tolerance of rhizobacteria from rice soil in Northern Thailand

Plant growth-promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. A total of 216 phosphate-solubilizing bacterial isolates were isolated from different rice rhizospheric soil in Northern Thailand. These isolate were screened in vitro for their plant growth-promoting activities such as solubilization of inorganic phosphate, ammonia (NH₃), catalase and cell wall-degrading enzyme activity. It was found that 100% solubilized inorganic phosphate, 77.77% produced NH₃ and most of the isolates were positive for catalase. In addition, some strains also produced cell wall-degrading enzymes such as protease (7%), chitinase (1%), cellulase (3%) and β-glucanase (3%), as evidenced by phenotypic biochemical test and quantitative assay using spectrophotometry. The isolates could exhibit more than two or three plant growth-promoting (PGP) traits, which may promote plant growth directly or indirectly or synergistically. Part of this study focused on the effect of NaCl, temperature, and pH on a specific the bacterial isolate Acinetobacter CR 1.8. Strain CR 1.8 was able to grow on up to 25% NaCl, between 25 and 55°C, and at pH 5–9. Maximum solubilization of tricalcium phosphate and aluminium phosphate was obtained at neutral pH, and 37°C. Strain CR 1.8 had protease activity but no cellulase, β-glucanase and cellulase activities.