Characterization and monitoring of selected rhizobial strains isolated from tree legumes in Thailand

Tree legume rhizobia were isolated from five tree legumes; Acacia auriculaformis Cunn., A. mangium Willd., Milletia leucantha Kurz., Pterocarpus indicus Willd., and Xylia xylocarpa Taub. grown in Thailand.Forty four highly effective rhizobial strains were selected on the basis of nitrogenase activity, number of nodules and plant biomass. The selected strains were characterized in both terms of physiology andgenetics. Most of the strains are slow grower and able to nodulate cowpea rather than soybean. In addition, IAA production could be detected only from few strains. When almost complete 16S rRNAsequences were analysed, the results indicated that most of the selected strains most likely belong to Bradyrhizobium elkanii and Bradyrhizobium sp. except strains AA67 and PT59 which most likely belong to B. japonicum. The nodule occupancy of selected strains in forest soil condition was investigated by using GUS reporter gene. The nodule occupancy is in the range of 63 - 100%. This suggests theappropriate strains should be produced as inoculum for further application in reforestation programmes in Thailand

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2-fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade

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

Regulation of nitrogen fixation in Bradyrhizobium sp. strain DOA9 involves two distinct NifA regulatory proteins that are functionally redundant during symbiosis but not during free-living growth

The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (beta-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc

Arbuscular mycorrhizal status of indigenous tree species used to restore seasonally dry tropical forest in northern Thailand

Arbuscular Mycorrhizal (AM) status of native plants in the tropical forest of northern Thailand was surveyed. Twenty four framework tree species, used to forest restoration were examined at 3 sites: FORRU=s research tree Nursery (FN), Forest Restoration plot (FR) and Natural Forest (NF). Eleven dominant herb species were examined at 2 sites: Degraded Watershed (DW) and Forest Soil extraction area (FS). Rhizosphere soil samples were collected and AM fungal spores were counted and identified morphologically. Most plant species were intensively colonized by AM fungi except Cyperus cyperoides. Twenty four AM species were identified: Glomus (15 species), Acaulospora (6 species) and Scutellospora (3 species). Glomus rubiforme was the dominant species. Spore density varied from 16.1 to 97.4 per 100 g soil (averaged 59.7). Spore number at DW and FS were 129 and 479 spores, respectively, with species richness of 6 and 8, respectively. Spore number at FN, FR and NF were 1,152, 2,337 and 1,376 spores, respectively, with species richness of 17, 21 and 15, respectively. The AM diversity was lower in the sites dominated by herbs than in sites examined for trees. In the deforested sites, reduced plant diversity was related with reduced mycorrhizal diversity. In contrast, the trial plot had the highest AM fungal community. Therefore, the forest restoration techniques allow tree species grown in nursery to become AM associated. The association is still maintained after planting out trees in restored area