Isamu Maeda

Purple non-sulfur bacteria (PNSB) have attracted attention as a group of microorganisms with plant growth-promoting abilities. Notably, agriculturally important PNSB, including members of the genera Rhodopseudomonas and Rhodobacter, are classified within the class α-Proteobacteria. However, molecular visualization of these bacteria in plant tissues remains challenging without bacterial genetic manipulation. In this study, a DIG-labeled RNA probe was developed from a 16S rRNA region showing relatively high sequence conservation among the tested α-Proteobacterial strains. Northern blot analysis using RNA extracted from 13 bacterial strains demonstrated preferential hybridization of the probe to the tested α-Proteobacterial strains. Then, in situ hybridization (ISH) of fixed bacterial cells produced positive signals only in the tested α-Proteobacterial strains. Finally, after inoculation of Rhodopseudomonas palustris C2 during rice seed priming and seedling hydroponic cultivation, ISH analysis of rice roots revealed probe-positive bacterial structures in root epidermal and root hair-associated regions. Collectively, these results demonstrate the applicability of the developed RNA probe for molecular visualization from extracted RNA and bacterial cells to rice root tissues under controlled inoculation conditions and provide a useful approach for investigating bacterial localization and plant–bacteria interactions in rice roots.

In rice, symbiotic N₂ fixation via nodule bacteroids does not take place naturally. Although N₂ fixation by endophytic and associative diazotrophs has been reported in rice, the main organs and seasonal regulation for the N₂ fixation have not been elucidated. In this study, seasonal changes in nitrogenase (acetylene reduction) activity and carbohydrate contents in elongated culm (EC), unelongated stem (US), and crown root (CR) were investigated in manure compost (MC)- and chemical fertilizer (CF)-applied rice. Nitrogenase activity increased after rooting (June) and reached the highest activity in US of MC-applied rice at panicle initiation (August). The sucrose content in EC continued to increase after rooting regardless of the applied materials, whereas the glucose content in US increased after rooting only in CF-applied rice, suggesting higher consumption of glucose in US of MC-applied rice. There were significant differences among bacterial microbiota in EC, US, and CR at panicle initiation. In addition, Clostridia class anaerobes were more abundant in US of MC-applied rice than in EC and CR of MC-applied rice. Such difference was not observed in US of CF-applied rice. These results suggest the suitability of US of MC-applied rice at panicle initiation as a site of N₂ fixation under anaerobic conditions.

In rice, symbiotic N fixation via nodule bacteroids does not take place naturally. Although N fixation by endophytic and associative diazotrophs has been reported in rice, main organs and seasonal regulation for the N fixation have not been elucidated. In this study, therefore, seasonal changes of nitrogenase (acetylene reduction) activity and carbohydrate contents in culm internode (IN), basal nodes (BN), and crown root (CR) were investigated in manure compost (MC)- and chemical fertilizer-applied rice. Nitrogenase activity increased after rooting, and reached the highest activity in BN of MC-applied rice at panicle initiation. In BN of MC-applied rice, the sucrose content was low whereas the higher glucose content was detected at panicle initiation. Although bacterial and diazotrophic bacterial populations were most abundant in CR, the nitrogenase activity seemed to be restricted by its trace sucrose and glucose contents. In addition, alpha-Proteobacteria and Clostridia phyla were abundant in BN-specific bacterial microbiota at panicle initiation. These results suggest that the increased N fixation in BN at panicle initiation might be sustained both by the non-structural carbohydrate supply from photosynthetic apparatus and BN-specific bacterial microbiota including diazotrophs.

Biological nitrogen fixation complements nitrogen from fertilizers in crop plants under natural conditions. It also contributes to the reduction in chemical fertilizer (CF) utilization in cultivated lands, which fits the concept of sustainable agriculture. From this viewpoint, however, it is still unknown in paddy fields how soil bacterial nitrogenase and microbiota are affected by applied materials in the soil. Therefore, in this study, the effects of long-term material applications on potential nitrogenase activity and the microbiota of soil bacteria were investigated. The nitrogenase activity tended to be higher in manure compost (MC)-applied soils than in CF-applied soils in both summer and winter. Soil bacterial alpha diversity increased whereas soil ammonia availability decreased with the MC application. The dynamic response of soil bacterial microbiota was caused by the MC application. The abundance of Nitrospira, a class of ammonia and nitrite oxidation bacteria, was lower and the abundance of alpha-Proteobacteria was higher in the MC-applied soils than in the CF-applied soils. These results suggest that the alpha diversity increase and restricted availability of NH₃-N might contribute to the increase in potential nitrogenase activity in the long-term MC-applied soils.

Lead (Pb) pollution from local mines and industrial use increases risks for human, animal, and plant health. Pectin is an effective chelator of Pb, and it has been shown that a unicellular green alga, Penium margaritaceum, synthesizes pectin in the cell wall. In this study, the ability of P. margaritaceum to remove Pb from an aqueous solution was investigated. Energy dispersive X-ray spectroscopy revealed that two strains of P. margaritaceum accumulated Pb on the cell surface. Hence, P. margaritaceum cells were immobilized on cellulose filter paper. The immobilized algal cells were soaked in 1.0 mg/L Pb solution with gentle shaking for 8 h, and Pb in the solution and on the filter paper was measured by flame atomic absorption spectrometry. The immobilized algal cells continuously decreased the Pb concentration to less than 0.5 mg/L and recovered 31.8–32.7% of added Pb. The specific decrease in Pb and increase in Ca were observed in the presence of 1.0 mg/L each of Ca, Mg, Na, and K. Fourier transform infrared spectra suggested that the carboxylic acid group would be responsible for the adsorption of Pb. This study is the first to demonstrate the effectiveness of the immobilized P. margaritaceum cell in removing Pb from aqueous solutions with simple solid–liquid separation.

Biological nitrogen fixation catalyzed by Mo-nitrogenase of symbiotic diazotrophs has attracted interest because its potential to supply plant-available nitrogen offers an alternative way of using chemical fertilizers for sustainable agriculture. Phototrophic purple nonsulfur bacteria (PNSB) diazotrophically grow under light anaerobic conditions and can be isolated from photic and microaerobic zones of rice fields. Therefore, PNSB as asymbiotic diazotrophs contribute to nitrogen fixation in rice fields. An attempt to measure nitrogen in the oxidized surface layer of paddy soil estimates that approximately 6–8 kg N/ha/year might be accumulated by phototrophic microorganisms. Species of PNSB possess one of or both alternative nitrogenases, V-nitrogenase and Fe-nitrogenase, which are found in asymbiotic diazotrophs, in addition to Mo-nitrogenase. The regulatory networks control nitrogenase activity in response to ammonium, molecular oxygen, and light irradiation. Laboratory and field studies have revealed effectiveness of PNSB inoculation to rice cultures on increases of nitrogen gain, plant growth, and/or grain yield. In this review, properties of the nitrogenase isozymes and regulation of nitrogenase activities in PNSB are described, and research challenges and potential of PNSB inoculation to rice cultures are discussed from a viewpoint of their applications as nitrogen biofertilizer.

Aquaponics is a circulating and sustainable system that combines aquaculture and hydroponics and forms a symbiotic relationship between fish, plants, and microorganisms. We hypothesized that feed alone could support plant growth, but the symbiosis with fish adds some beneficial effects on plant growth in aquaponics. In this study, we created three closed culture systems, namely, aquaponics, hydroponics without nitrogen (N) and phosphorus (P), and aquaculture, and added the same amount of feed containing N and P to all the treatments in order to test the hypothesis. Accumulation of NO₃⁻ and PO₄³⁻ was alleviated in aquaponics and hydroponics as a result of plant uptake. Lettuce plants grown in aquaponics grew vigorously until 2 weeks and contained a constant level of N in plants throughout the production period, whereas those in hydroponics grew slowly in the early stage and then vigorously after 2 weeks with a late increment of N concentration. These results suggest that catfish help with the faster decomposition of the feed, but, in hydroponics, feed can be slowly dissolved and decomposed owing to the absence of the fish. The bacterial community structures of the culture solution were investigated using 16S rRNA gene amplicon sequencing. At the class level, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria were the major microbial groups in the solutions. Aquaponics prevented the pollution of tank solution and maintained a higher water quality compared with hydroponics and aquaculture, suggesting that aquaponics is a more sustainable cultivation system even in a small-scale system.

Geraniol is one of the important aromatic ingredients in alcoholic beverages. Bioconversions of geraniol to other terpenoids and genes involved in the oxidation of geraniol were investigated. Geranic acid and citronellic acid were detected in yeast culture, where geraniol or nerol was added. Addition of citral, a mixture of geranial and neral, resulted in the production of geranic acid and citronellic acid, whereas the addition of citral or citronellal resulted in the production of citronellic acid, suggesting that citronellic acid might be produced through the conversion of citral to citronellal followed by the oxidation of citronellal. Consumption of geraniol and production of geranic acid, citronellic acid, and citronellol were affected in adh1Δ, adh3Δ, adh4Δ, and sfa1Δ yeast strains, which possess single deletion of a gene encoding alcohol dehydrogenase. This is the first report of the bioconversion of monoterpene alcohols, geraniol and nerol, to geranic acid and citronellic acid in yeast culture.

Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean. Tempe contains linoleic and α-linolenic acids, which are bactericidal against S. aureus and B. subtilis, and monolinolenins, which are bactericidal against B. subtilis.