Indigofera tinctoria leaf powder as a promising additive to improve indigo fermentation prepared with sukumo (composted Polygonum tinctorium leaves).

Being insoluble in the oxidize form, indigo dye must be solubilized by reduction for it to penetrate textile. One of the procedures is the reduction by natural bacterial fermentation. Sukumo, composted leaves of Polygonum tinctorium, is a natural source of indigo in Japan. Although sukumo has an intrinsic bacterial seed, the onset of indigo reduction with this material may vary greatly. Certain additives improve indigo fermentation. Here, we studied the effects of Indigofera tinctoria leaf powder (LP) on the initiation of indigo reduction, bacterial community, redox potential (ORP), and dyeing intensity in the initial stages and in aged fermentation fluids prepared with sukumo. I. tinctoria LP markedly decreased ORP at day 1 and stabilised it during early fermentation. These effects could be explained by the phytochemicals present in I. tinctoria LP that act as oxygen scavengers and electron mediators. Using next generation sequencing results, we observed differences in the bacterial community in sukumo fermentation treated with I. tinctoria LP, which was not influenced by the bacterial community in I. tinctoria LP per se. The concomitant decrease in Bacillaceae and increase in Proteinivoraceae at the onset of fermentation, increase in the ratio of facultative to obligate anaerobes (F/O ratio), or the total abundance of facultative anaerobes (F) or obligate anaerobes (O) (designated F + O) are vital for the initiation and maintenance of indigo reduction. Hence, I. tinctoria LP improved early indigo reduction by decreasing the ORP and hasten the appropriate transitions in the bacterial community in sukumo fermentation.

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia.

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.

Bacillus polygoni sp. nov., a moderately halophilic, non-motile obligate alkaliphile isolated from indigo balls.

A moderately halophilic, obligate alkaliphile (growth range pH 8-12), designated strain YN-1(T), was isolated from indigo balls obtained from Ibaraki, Japan. The cells of the isolate stained Gram-positive, and were aerobic, non-motile, sporulating rods which grew optimally at pH 9. The strain grew in 3-14% NaCl with optimum growth in 5% NaCl. It hydrolysed casein and Tweens 20, 40 and 60, but not gelatin, starch, DNA or pullulan. Its major isoprenoid quinone was MK-7 and its cellular fatty acid profile mainly consisted of anteiso-C(15:0), anteiso-C(17:0) and anteiso-C(17:1). 16S rRNA phylogeny suggested that strain YN-1(T) was a member of group 7 (alkaliphiles) of the genus Bacillus, with the closest relative being Bacillus clarkii DSM 8720(T) (similarity 99.5%). However, DNA-DNA hybridization showed a low DNA-DNA relatedness (7%) of strain YN-1(T) with B. clarkii DSM 8720(T). Owing to the significant differences in phenotypic and chemotaxonomic characteristics, and phylogenetic and DNA-DNA relatedness data, the isolate merits classification as a new species, for which the name Bacillus polygoni is proposed. The type strain of this species is YN-1(T) (=JCM 14604(T)=NCIMB 14282(T)).

Rhizobium indigoferae sp. nov. and Sinorhizobium kummerowiae sp. nov., respectively isolated from Indigofera spp. and Kummerowia stipulacea.

Forty-eight rhizobial isolates from root nodules of Indigofera and Kummerowia, two genera of annual or perennial wild legumes growing in the Loess Plateau in north-western China, were characterized by a polyphasic approach. Two main groups, cluster 1 and cluster 2, were defined based upon the results of numerical taxonomy, SDS-PAGE of whole-cell proteins and DNA relatedness. All the isolates within cluster 1 were isolated from Indigofera and they were identified as Rhizobium strains by 16S rRNA gene analysis. DNA relatedness of 29.5-48.9% was obtained among the cluster 1 isolates and the reference strains for defined Rhizobium species. Cluster 2 consisted of isolates from Kummerowia stipulacea and was identified as belonging to Sinorhizobium by 16S rRNA gene analyses. DNA relatedness varied from 5.2 to 41.7% among the isolates of cluster 2 and reference strains for Sinorhizobium species. Considering the existence of distinctive features among these two groups and related species within the genera Rhizobium and Sinorhizobium, we propose two novel species, Rhizobium indigoferae sp. nov. for cluster 1, with isolate CCBAU 71714(T) (= AS 1.3046(T)) as the type strain, and Sinorhizobium kummerowiae sp. nov. for cluster 2, with isolate CCBAU 71042(T) (= AS 1.3045(T)) as the type strain.

[A study on taxonomy of rhizobia isolated from Kummerwia sp. and Indigofera sp].

The rhizobial strains isolated from Kummerowia and Indigofera and the known reference strains were classified by performing numerical taxonomy. New isolated strains were divided into two new clusters at 83% similarity level. Based on the numerical taxonomy, additional isolates in each cluster were studied by using SDS-PAGE of whole-cell protein. Twenty-four strains isolated from Kummerowia fell into cluster 1. Twenty strains isolated from Indigofera fell into cluster 2. The results of G + C mol% and DNA homology analysis showed that the DNA homologies between the central strain SH713 and SHL042 and the 13 type strains were less than 61%. Thus, the rhizobial strains from Kummerowia and Indigofera were two new individual species of Rhizobium.