Molecular and phenotypic characterization of potential plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> from rice and maize rhizospheres

In this study, Pseudomonas species were isolated from the rhizospheres of two plant hosts: rice (Oryza sativa cultivar Pathum Thani 1) and maize (Zea mays cultivar DK888). The genotypic diversity of isolates was determined on basis of amplified rDNA restriction analysis (ARDRA). This analysis showed that both plant varieties selected for two distinct populations of Pseudomonas. The actual biocontrol and plant promotion abilities of these strains was confirmed by bioassays on fungal (Verticillum sp., Rhizoctonia solani and Fusarium sp.) and bacterial (Ralstonia solanacearum and Bacillus subtilis) plant pathogens, as well as indole-3-acetic acid (IAA) production and carbon source utilization. There was a significant difference between isolates from rice and maize rhizosphere in terms of biological control against R.  solanacearum and B.  subtilis. Interestingly, none of the pseudomonads isolated from maize rhizosphere showed antagonistic activity against R.  solanacearum. This study indicated that the percentage of pseudomonad isolates obtained from rice rhizosphere which showed the ability to produce fluorescent pigments was almost threefold higher than pseudomonad isolates obtained from maize rhizosphere. Furthermore, the biocontrol assay results indicated that pseudomonad isolated from rice showed a higher ability to control bacterial and fungal root pathogens than pseudomonad isolates obtained from maize. This work clearly identified a number of isolates with potential for use as plant growth-promoting and biocontrol agents on rice and maize.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol–chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.     

Soybean cultivars affect nodulation competition of Bradyrhizobium japonicum strains

The influence of five Thai soybean cultivars on nodulation competitiveness of four Bradyrhizobium japonicum strains was investigated. Cultures of B. japonicum strains THA5, THA6, USDA110 and SEMIA5019 were mixed with each other prior to inoculating germinated soybean seeds growing in Leonard jars with nitrogen-free nutrient solution. At harvest, nodule occupancy by each strain was determined by a fluorescent antibody technique. The term ‘general competitive ability’ was introduced to describe the average competitive nodule occupancy of a strain in paired co-inoculation with a number of strains on soybean. The nodule occupancies by an individual strain were directly correlated with the proportions of that strain in the inoculum mixtures. USDA110 showed higher nodulation competitiveness than the other strains on three of the five cultivars. The Thai strain THA6 appeared to be more competitive than USDA110 on cultivar SJ5. Thus, nodulation competitiveness of the B. japonicum strains was affected by the cultivars of soybean used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Detection of Bradyrhizobium spp. and B. japonicum in Thailand by primer-based technology and direct DNA extraction

Total chromosomal DNAs from 20 Bradyrhizobium spp. strains (10 strains isolated from Vigna radiata and 10 from Arachis hypogaea) and 18 B. japonicum strains isolated from Glycine max were extracted. These DNAs served as templates for REP, ERIC and RAPD primers in PCR analyses. The patterns of the resulting PCR products were analyzed and highly specific for each strain, especially when grouped together with their antibiotic-resistance profiles. A method for extracting DNA directly from soil was developed. Recovery was approximately 30 g DNA g-1 soil and the procedure yielded DNA suitable for PCR amplification.     

Comparative study of endophytic and endophytic diazotrophic bacterial communities across rice landraces grown in the highlands of northern Thailand

Communities of bacterial endophytes within the rice landraces cultivated in the highlands of northern Thailand were studied using fingerprinting data of 16S rRNA and nifH genes profiling by polymerase chain reaction–denaturing gradient gel electrophoresis. The bacterial communities’ richness, diversity index, evenness, and stability were varied depending on the plant tissues, stages of growth, and rice cultivars. These indices for the endophytic diazotrophic bacteria within the landrace rice Bue Wah Bo were significantly the lowest. The endophytic bacteria revealed greater diversity by cluster analysis with seven clusters compared to the endophytic diazotrophic bacteria (three clusters). Principal component analysis suggested that the endophytic bacteria showed that the community structures across the rice landraces had a higher stability than those of the endophytic diazotrophic bacteria. Uncultured bacteria were found dominantly in both bacterial communities, while higher generic varieties were observed in the endophytic diazotrophic bacterial community. These differences in bacterial communities might be influenced either by genetic variation in the rice landraces or the rice cultivation system, where the nitrogen input affects the endophytic diazotrophic bacterial community.     

Characterization of Arbuscular Mycorrhizal Fungus Communities of Aquilaria crassna and Tectona grandis Roots and Soils in Thailand Plantations

Aquilaria crassna Pierre ex Lec. and Tectona grandis Linn.f. are sources of resin-suffused agarwood and teak timber, respectively. This study investigated arbuscular mycorrhizal (AM) fungus community structure in roots and rhizosphere soils of A. crassna and T. grandis from plantations in Thailand to understand whether AM fungal communities present in roots and rhizosphere soils vary with host plant species and study sites. Terminal restriction fragment length polymorphism complemented with clone libraries revealed that AM fungal community composition in A. crassna and T. grandis were similar. A total of 38 distinct terminal restriction fragments (TRFs) were found, 31 of which were shared between A. crassna and T. grandis. AM fungal communities in T. grandis samples from different sites were similar, as were those in A. crassna. The estimated average minimum numbers of AM fungal taxa per sample in roots and soils of T. grandis were at least 1.89 vs. 2.55, respectively, and those of A. crassna were 2.85 vs. 2.33 respectively. The TRFs were attributed to Claroideoglomeraceae, Diversisporaceae, Gigasporaceae and Glomeraceae. The Glomeraceae were found to be common in all study sites. Specific AM taxa in roots and soils of T. grandis and A. crassna were not affected by host plant species and sample source (root vs. soil) but affected by collecting site. Future inoculum production and utilization efforts can be directed toward the identified symbiotic associates of these valuable tree species to enhance reforestation efforts.     

Diversity of nitrogen-fixing cyanobacteria under various ecosystems of Thailand: population dynamics as affected by environmental factors

Investigation of N₂-fixing cyanobacteria from Thai soil was carried out at 2-month intervals between July 1997 and November 1999 to determine the population number, population dynamics and favourable habitats. Sites were selected in three parts of Thailand; North, Central and Northeast. In each part, various soil ecosystems were used as sampling sites; at highest elevation as on the top of the mountain, in the middle and at the foot of the mountain, as well as in flat areas of agricultural practice and uncultivated areas. Generally, a high population of N₂-fixing cyanobacteria was found in agricultural areas where rice cultivation was practised, rather than in other sites. The population dynamics in the mountain and uncultivated areas were less fluctuating than in agricultural areas. The population densities in agricultural areas increased in the rainy season and decreased during the dry season. Other environmental factors such as temperature, moisture and pH also affected the population densities in different habitats. Cyanobacterial diversity was notably influenced by the type of ecosystem in both dry and rainy seasons. The cultivation area containing rice in rotation with other crops contained the most genetically diverse range of species.     

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.