Neelawan Pongsilp

CO-INOCULATION OF SOYBEAN (GLYCINE MAX) WITH ACTINOMYCETES AND BRADYRHIZOBIUM JAPONICUM ENHANCES PLANT GROWTH, NITROGENASE ACTIVITY AND PLANT NUTRITION

The aim of this study was to determine the potential of the endophytic actinomycetes that produce plant growth promoters used as co-inoculants with Bradyrhizobium japonicum to promote the growth of soybean. These endophytes exhibited the potential to enhance plant growth, nitrogenase activity of root nodules and plant nutrient uptake. Co-inoculum of B. japonicum with Nocardia alba conferred the maximum yield of root and shoot dry weight. All single-inoculated actinomycetes strains had the ability to enhance plant growth. Noc. alba and Nonomuraea rubra increased total plant dry weight up to 2.14-fold and 2.11-fold, respectively, when compared to the uninoculated controls. Co-inoculations of B. japonicum with each of Noc. alba, Non. Rubra, and Actinomadura glauciflava increased acetylene reduction activity up to 1.7 to 2.7-fold. For plant mineral composition, all of co-inoculation treatments significantly increased the nutrient levels of nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe) and zinc (Zn) within a soybean plant.

Impact of rhizobial inoculants on rhizosphere bacterial communities of three medicinal legumes assessed by denaturing gradient gel electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis (DGGE) was used to study the impact of rhizobial inoculants on the rhizosphere bacterial communities of three medicinal legumes: Indigofera tinctoria, Pueraria mirifica and Derris elliptica Benth. Rhizosphere soils were collected from these legumes grown naturally in 11 provinces of Thailand. The host-specific rhizobial strains were inoculated to their hosts planted in the collected rhizosphere soils of each legume. Four months after planting, total bacterial communities DNA was extracted from the uninoculated rhizosphere soils and the inoculated rhizosphere soils. DGGE fingerprints of PCR-amplified 16S rDNA were obtained from the bacterial communities. PCR-DGGE analysis showed that the bacterial community structures in native rhizospheres of the three legumes were different from each other based on the generated dendrogram and Sorensen’s index. These results suggest that different plant species and soil characteristics synergically affected the rhizosphere bacterial communities. The bacterial diversity of I. tinctoria and P. mirifica native rhizospheres were significantly different from that of D. elliptica Benth. native rhizosphere. Our results also showed that the inoculants contributed to the slight changes in rhizosphere community structures. In comparison with each other, the plants appeared to have a much stronger influence on the bacterial communities rather than the inoculants. Hierarchical cluster analysis revealed that the community structure of the inoculated rhizosphere of D. elliptica Benth. was more divergent from those of inoculated rhizospheres of I. tinctoria and P. mirifica. The ribotype richness which indicates species diversity, was highest in I. tinctoria rhizosphere, followed by P. mirifica rhizosphere and D. elliptica Benth. rhizosphere, respectively.

Characterization of root-nodule bacteria isolated from the medicinal legume Indigofera tinctoria

Fourteen root-nodule bacteria isolated from the medicinal legume Indigofera tinctoria were characterized for their phenotypic features including growth curves, utilization of carbon and nitrogen sources, antibiotic resistance, vitamin requirement and growth under different conditions. The partial sequences of the 16S rDNA of these strains were obtained and BLASTN analysis revealed that the microsymbionts of I. tinctoria were related to members of five distinct genera: Rhizobium, Sinorhizobium, Bradyrhizobium, Cupriavidus and Pseudoalteromonas. The partial nifH gene of Pseudoalteromonas-like strain DASA 57075 had 96% similarity with nifH genes of members of Bradyrhizobium. The partial nodC gene of Pseudoalteromonas-like strain DASA 57075 showed 88% similarity with the nodC gene of several rhizobia including Sinorhizobium, Bradyrhizobium and Mesorhizobium. We propose a bacterium that is related to Pseudoalteromonas from the gamma-class of Proteobacteria as a new legume symbiont. This is also the first report that the same species of legume can be nodulated by bacteria from up to five different genera in three distinct classes.

Genotypic diversity among rhizospheric bacteria of three legumes assessed by cultivation-dependent and cultivation-independent techniques.

The genotypic diversity of rhizospheric bacteria of 3 legumes including Vigna radiata, Arachis hypogaea and Acacia mangium was compared by using cultivation-dependent and cultivation-independent methods. For cultivation-dependent method, Random amplified polymorphic DNA (RAPD) profiles revealed that the bacterial genetic diversity of V. radiata and A. mangium rhizospheres was higher than that of A. hypogaea rhizosphere. For cultivation-independent method, Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes revealed the difference in bacterial community and diversity of rhizospheres collected from 3 legumes. The ribotype richness which indicates species diversity, was highest in V. radiata rhizosphere, followed by A. hypogaea and A. mangium rhizospheres, respectively. Three kinds of media were used to cultivate different target groups of bacteria. The result indicates that the communities of cultivable bacteria in 3 rhizospheres recovered from nutrient agar (NA) medium were mostly different from each other, while Bradyrhizobium selective medium (BJSM) and nitrogen-free medium shaped the communities of cultivable bacteria. Nine isolates grown on BJSM were identified by 16S rRNA gene sequence analysis. These isolates were very closely related (with 96% to 99% identities) to either one of the three groups including Cupriavidus-Ralstonia group, Bacillus group and Bradyrhizobium-Bosea-Afipia group. The rhizospheres were also examined for their enzymatic patterns. Of 19 enzymes tested, 3 rhizospheres were distinguishable by the presence or the absence of leucine acrylamidase and acid phosphatase. The selected cultivable bacteria recovered from NA varied in their abilities to produce indole-acetic acid and ammnonia. The resistance to 10 antibiotics was indistinguishable among bacteria isolated from different rhizospheres.

Heavy metal tolerant Metalliresistens boonkerdii gen. nov., sp. nov., a new genus in the family Bradyrhizobiaceae isolated from soil in Thailand

Bacterial strains from inoculated soybean field soil in Thailand were directly isolated using Bradyrhizobium japonicum selective medium (BJSM), on the basis of Zn(2+) and Co(2+) resistance of B. japonicum and B. elkanii. The isolates were classified into symbiotic and non-symbiotic groups by inoculation assays and Southern hybridization of nod and nif genes. In this study, a nearly full-length 16S rRNA gene sequence showed that the non-symbiotic isolates were more closely related to members of Rhodopseudomonas and to a number of uncultured bacterial clones than to members of Bradyrhizobium. Therefore, a polyphasic study was performed to determine the taxonomic positions of four representatives of the non-symbiotic isolates. Multilocus phylogenetic analysis of individual genes and a combination of the 16S rRNA and three housekeeping genes (atpD, recA and glnII) supported the placement of the non-symbiotic isolates in a different genus. The ability of heavy metal resistance in conjunction with phenotypic analyses, including cellular fatty acid content and biochemical characteristics, showed that the non-symbiotic isolates were differentiated from the other related genera in the family Bradyrhizobiaceae. Therefore, the non-symbiotic isolates represented a novel genus and species, for which the name Metalliresistens boonkerdii gen. nov., sp. nov. is proposed. The type strain is NS23 (= NBRC 106595(T)=BCC 40155(T)).

Diversity and antibiotic resistance patterns of enterobacteria isolated from seafood in Thailand

ABSTRACT Contamination with enterobacteria was detectable in 89% of seafood samples from three central seafood markets in Thailand. The average numbers obtained from the same type of seafood were between 1.3 ± 0.9 and 4.5 ± 1.3 log CFU/g per sample. Eighty-one strains and 16 species were distinguished based on ERIC-PCR patterns and TP-RAPD patterns, respectively. The highest prevalence (90% of strains) was resistant to penicillin G whereas none was resistant to gentamycin. In addition, 63% exhibited multidrug resistance. The 16S rDNA sequences of a representative strain from each species exhibited 99% identity to either one of six genera including Citrobacter, Enterobacter, Klebsiella, Providencia, Serratia, and Yersinia. Three β-lactamase genes including blaTEM, ampC, and shv were detected at the frequencies of 43%, 27%, and 24%, respectively. The representative strains possessing β-lactamase genes exhibited β-lactamase activity ranging from 1.96 ± 0.88 to 11.3 ± 0.37 μmol of hydrolyzed nitrocefin/min/mg protein.