Improving the reliability of a metagenomic sequencing ana­lysis requires the use of control samples, known as mock communities. Therefore, mock communities must be prepared with high accuracy and reproducibility, which is particularly challenging for cellular mock communities. In the present study, we prepared a cellular mock community consisting of bacterial strains representative of the human and surrounding environmental microbiomes to demonstrate the suitability of a HPLC-based method that measures the genome number of cells. This method proved to be more accurate and reproducible for preparing cellular mock communities than traditional cell counting-based enumeration methods.

Digital PCR is a technique that quantifies target genes based on the absence or presence of the targets in PCR amplicons. The present study exami­ned group-specific probes for the quantification of mcrA genes in six methanogenic archaeal groups and “Candidatus Methanoperedens” by digital PCR with the universal primers ML-f and ML-r. A digital PCR ana­lysis of paddy field soil detected all the targets, with the dominant and minor groups being Methanomicrobiales and Methanobrevibacter spp., respectively (10⁷ and 10⁴ copies [g dry soil]^–1). This method has the potential to reveal the dynamics of specific methanogenic archaeal groups in the environment.

Nitrous oxide (N₂O) is a key atmospheric greenhouse gas that contributes to global warming, with anthropogenic N₂O emissions from agriculture being a particular concern. Among agricultural sources, unknown soil organisms in the legume rhizosphere emit N₂O from degraded root nodules. To discriminate between fungal and bacterial N₂O emissions, we adopted an isotopomer ana­lysis, which provides site preference values (the difference in ¹⁵N abundance of the central and terminal N atoms in the N₂O molecule). The addition of nitrite instead of nitrate to soybean nodulated roots significantly increased SP_N2O from –3.5‰ to 4.2‰ in a pot system. Moreover, a mutation of the nirK gene (encoding dissimilatory nitrite reductase) in symbiotic bradyrhizobia significantly increased SP_N2O from 4.2‰ to 13.9‰ with nitrite. These results suggest that nitrite-utilizing N₂O emissions via fungal denitrification occurred in the model pot system of the soybean rhizosphere. Microscopic observations showed fungal hyphae and crescent spores around N₂O-emitting nodules. Therefore, we isolated single spores from soybean nodules under a microscope. A phylogenetic ana­lysis revealed that all 12 fungal isolates were Fusarium species, which exist in soybean field soil. When these isolates were cultivated in glycerol-peptone medium supplemented with nitrate or nitrite (1‍ ‍mM), 11 of the 12 isolates strongly converted nitrite to N₂O; however, no N₂O emissions were noted in the presence of nitrate. A ¹⁵N-nitrite tracer experiment revealed that one N₂O molecule was derived exclusively from two molecules of nitrite (NO₂^–) in the fungal culture. These results suggest that nitrite-utilizing Fusarium fungi mediate N₂O emissions in the soybean rhizosphere.

The whitefly, Bemisia tabaci, is a notorious insect pest that transmits plant pathogenic viruses to a wide range of economically important crops. An invasive genetic group of B. tabaci, Mediterranean Q2 (MED Q2), has recently spread to Europe, USA, and Asia. In the present study, we investigated the prevalence of MED Q2 in Japanese agricultural sites and found that its distribution has expanded since it was initially detected in 2013. A polymerase chain reaction ana­lysis revealed that all MED Q2 individuals were infected with Rickettsia. Rickettsia titers increased during nymphal development, presumably in response to the nutritional needs of the host. A fluorescence in situ hybridization ana­lysis revealed that Rickettsia was densely located near Portiera-containing bacteriocytes at all growth stages. Therefore, Rickettsia may play an important role, such as supplying nutrients to the host, in cooperation with Portiera. Transfer experiments indicated that MED Q2 was as effective a vector for Tomato yellow leaf curl virus as MED Q1 and, thus, is a high-risk agricultural pest. These results provide important insights into the biology and ecology of invasive MED Q2 to effectively control its spread and minimize its impact on crops.

Phytophthora root and stem rot (PRSR) caused by Phytophthora sojae is a major concern for global soybean production. To identify a bacterial biocontrol agent against PRSR, 73 rhizobacterial strains were isolated from wild and cultivated legumes and screened for their protective activities against PRSR in pot experiments. Strain GVv1 was selected for its consistent protective effect through repeated pot experiments. The protective effect of this strain was similar to that of the fungicide mancozeb-metalaxyl. A dual-culture assay showed that GVv1 produced antifungal metabolites effective against P. sojae. To evaluate the potential adaptability of GVv1 to the soybean rhizosphere environment, its growth was exami­ned in soybean root exudates and nutrient medium, both supplemented with daidzein, an antimicrobial isoflavone secreted by soybean roots. GVv1 proliferated using soybean root exudates and had sufficient tolerance to daidzein to colonize the soybean rhizosphere. The plant growth-promoting effect of GVv1 on soybean plants was also investigated. GVv1 significantly increased shoot and root dry weights, indicating its plant growth-promoting activity. In vitro assays showed that GVv1 produced indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylate deaminase and solubilized insoluble phosphates. A taxonogenomic ana­lysis of the draft genome identified GVv1 as Enterobacter pseudoroggenkampii with high similarity (98.32% average nucleotide identity) to E. pseudoroggenkampii strain 155092^T. To the best of our knowledge, this is the first study to report the biocontrol and plant growth-promoting activities of E. pseudoroggenkampii.

Microbial community structures in mesophilic and low-temperature anammox and partial nitrification–anammox reactors were exami­ned by a 16S rRNA–gene amplicon sequencing ana­lysis. The anammox bacterium, Jettenia sp., was dominant, and nitrifying bacteria, including Nitrosomonas sp. (aerobic ammonia–oxidizing bacterium) and Nitrospira sp., (nitrite–oxidizing bacterium) coexisted in the bioreactors. Core coexisting bacteria, such as Sulfurisoma sp. and Zeimonas sp., showed oxygen-scavenging and NO₃^– reduction potentials. Sulfurisoma-related bacteria are distributed across wastewater treatment plants worldwide, particularly in denitrification systems. These results underscore the ecological and functional importance of microbial consortia in enhancing nitrogen removal efficiency.