Changes in the Endophytic Bacterial Community of Brassica rapa after Application of Systemic Insecticides.

Insecticides not only control target pests but also adversely affect non-target communities including humans, animals, and microbial communities in host plants and soils. The effect of insecticides on non-target communities, especially endophytic bacterial communities, remains poorly understood. Two phases of treatments were conducted to compare the trends in endophytic bacterial response after insecticide application. Endophytic bacteria were isolated at 2 and 4 weeks after germination. Most insecticide treatments showed a declining trend in bacterial diversity and abundance, whereas an increasing trend was observed in the control. Therefore, insecticide use negatively affected non-target endophytic bacterial communities. Bacillus spp. was mostly dominant in the early stage in both insecticide treatment and control groups. Nevertheless, in the matured stage, mostly bacteria including Pseudomonas spp., Priestia spp. were dominant in groups treated with high insecticide concentrations. Therefore, plants can regulate and moderate their microbiome during their lifecycle depending on surrounding environmental conditions.

Characterization of the Non-rhizobial Bacterial Community in the Nodule-Associated Soils of Akebono Soybeans and Isolation of Antibiotic Producing Amycolatopsis spp.

The unique Akebono soybeans are cultivated in Minobu Town, Yamanashi Prefecture, Japan. The biogeography of Akebono soybeans and the microbial diversity associated with their root nodules remain unexplored. This study investigated the nodule-associated microbial community of Akebono soybeans using molecular techniques. The results showed that the family Bradyrhizobiaceae was dominant in soybeans obtained from Minobu town Imata (MI), Minobu town IItomi (MS), and Minobu town Hirase (MN). In contrast, members of the family Pseudonocardiaceae were isolated from the nodule samples from Minobu town Yasaiku (MY). The community structure of MY was different from that of the other sites and the genus Amycolatopsis was dominant. The bacterial community in the nodule associated soil obtained from the Minobu area was different from that of soybeans cultivated in Kofu City (approximately 35 km away from Minobu Town). The MY1 strain of Amycolatopsis spp. was isolated from the nodule associated soil in MY and was found to produce antibiotics. This study showed that the bacterial community in the nodules and the adjacent rhizosphere may be a regional characteristic of Akebono soybeans and the specific Amycolatopsis spp. dominant in the nodule associated soil of MY was implicated in determining the bacterial community structure. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-00999-x.

Isolation of a Novel Endophytic Bacillus Strain Capable of Transforming Pentachlorophenol and Structure Determination of Pentachlorophenol Phosphate Using Single-Crystal X-ray Diffraction.

A novel approach for the remediation of upland soils contaminated with pentachlorophenol (C6HCl5O; PCP) (1), a fungicide, wood perservative, and herbicide, through the exploitation of plant-endophytic bacteria may overcome the existing issues in bioaugmentaion and phytoremidiation. In this study, we isolated the endophytic Bacillus sp. strain PCP15 and determined its metabolite of PCP (1). This strain degraded 8.03 µmol L-1 PCP (1) within 24 h and generated the novel metabolite PCP phosphate (3). The PCP15 strain showed nearly complete growth inhibition of 20 µmol L-1 PCP (1). In contrast, PCP15 showed resistance to PCP phosphate (3), indicating that the phosphorylation of PCP, which has never previously been reported in organisms, contributed to the detoxification of PCP (1) in bacterial cells. Our results show the potential for practical application of this strain in hybrid remediation of PCP (1)-contaminated soils and reveal a novel PCP (1) detoxification mechanism in organisms.

Green manure incorporation accelerates enzyme activity, plant growth, and changes in the fungal community of soil.

Green manure can sustain agricultural production, preserve biodiversity, and mitigate soil degradation caused by long-term application of chemical fertilizers. Moreover, the application of green manure can improve soil health through increased soil biological activities. Nevertheless, little attention has been paid to the effects of leguminous and non-leguminous plants on phosphorus- and carbon-related enzyme activities and fungal community composition in soil. In this study, a pot experiment was carried out to elucidate the effects of two green manures on plant growth promoting potential, phosphorus- and carbon-related enzyme activities, and soil fungal community composition. Two green manure treatments (Brassica juncea and hairy vetch), poultry compost and control (no amendment) were applied and soil samples were collected after incorporation of green manure and after plant harvest. The results revealed that plant growth with hairy vetch was significantly higher than that with B. juncea and poultry compost, and soil enzyme activities were markedly higher with hairy vetch than with B. juncea. Both green manure amendments altered the soil fungal community composition. It is possible that the incorporation of green manure into soil and their mineralization and decomposition were controlled by the carbon: nitrogen ratio of the manures and that these manures were easily degradable by soil fungi. In particular, the incorporation of leguminous (hairy vetch) green manure with a low carbon: nitrogen ratio resulted in better plant growth through fast mineralization. Our findings suggest that green manure incorporation is an effective practice and provides substantial benefits to the soil-plant system.

Metabolomics Analyses Reveal Metabolites Affected by Plant Growth-Promoting Endophytic Bacteria in Roots of the Halophyte Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum L. (common ice plant) is an edible halophyte. However, if ice plants are used to phytoremediate salinity soil, there are problems of slow initial growth, and a long period before active NaCl uptake occurs under higher salinity conditions. Application of endophytic bacteria may improve the problem, but there remain gaps in our understanding of how endophytic bacteria affect the growth and the biochemical and physiological characteristics of ice plants. The aims of this study were to identify growth-promoting endophytic bacteria from the roots of ice plants and to document the metabolomic response of ice plants after application of selected endophytic bacteria. Two plant growth-promoting endophytic bacteria were selected on the basis of their ability to promote ice plant growth. The two strains putatively identified as Microbacterium spp. and Streptomyces spp. significantly promoted ice plant growth, at 2-times and 2.5-times, respectively, compared with the control and also affected the metabolome of ice plants. The strain of Microbacterium spp. resulted in increased contents of metabolites related to the tricarboxylic acid cycle and photosynthesis. The effects of salt stress were alleviated in ice plants inoculated with the endobacterial strains, compared with uninoculated plants. A deeper understanding of the complex interplay among plant metabolites will be useful for developing microbe-assisted soil phytoremediation strategies, using Mesembryanthemum species.

Effect of co-application of Trichoderma spp. with organic composts on plant growth enhancement, soil enzymes and fungal community in soil.

The application of Trichoderma spp. has the potential to reduce not only mineral fertilizer use in agriculture but also improve soil health through increased soil biological activity. Trichoderma spp. have shown potential as bio-control agents and plant growth promoting ability, but little attention has been paid to the effect of Trichoderma spp. inoculation on nutrient availability and the soil microbiome. In this study, we evaluated the effect of Trichoderma spp. inoculation on nitrogen mineralization and quantified soil enzymatic activities along with plant growth promotion potential. The influence of Trichoderma spp. and organic amendments on the soil fungal community was also investigated. For this purpose, pots and incubation experiment was carried out, and seven treatments were set as follows; poultry compost (PC), poultry compost + RW309 (PCT), cattle compost (CC), cattle compost + RW309 (CCT), rapeseed oil cake (OC), inorganic fertilizer (N) and only soil (S) were set as control. We evaluated that Trichoderma sp. RW309 produced indole-3-acetic acid, which suggested that it could contribute to plant growth enhancement during early plant growth. Inoculation of RW309 with organic materials stimulated nitrogen mineralization and increased soil phosphatase activity. Furthermore, RW309 altered the fungal community in rhizosphere soil. However, cattle compost was a more suitable culture medium for RW309 than poultry compost in terms of nitrogen mineralization, soil enzyme activity, and growth of RW309. In conclusion, Trichoderma sp. RW309 could be considered for use as a bioorganic fertilizer in combination with organic compost to minimize the use of mineral fertilizers.

Non-target Impact of Dinotefuran and Azoxystrobin on Soil Bacterial Community and Nitrification.

Pesticides to protect crops from pests are subject to rigorous risk assessment before registration in Japan. However, further information needs to be collected regarding the assessment of impacts on the natural environment. In particular, nitrifying bacteria play a role in converting ammonium salts to nitrates in soil. However, there is limited research covering the effects of insecticides on nitrification, despite several fungicides and herbicides have an inhibitory effect on nitrifying bacteria. Therefore, we investigated the effect of pesticides on the nitrification when applied to soil. The application of both pesticides promoted ammonia oxidation, and suppressed nitrite oxidation in a high-concentration treatment of dinotefuran. In addition, it was clarified that the diversity and species richness of soil bacteria was significantly reduced when the pesticides were applied to the soil, and that the specific soil bacteria (Metyhlotenera spp.) dominated the application of the pesticides.

Biochemical characterization of NADH:FMN oxidoreductase HcbA3 from Nocardioides sp. PD653 in catalyzing aerobic HCB dechlorination.

Nocardioides sp. PD653 genes hcbA1, hcbA2, and hcbA3 encode enzymes that catalyze the oxidative dehalogenation of hexachlorobenzene (HCB), which is one of the most recalcitrant persistent organic pollutants (POPs). In this study, HcbA1, HcbA2, and HcbA3 were heterologously expressed and characterized. Among the flavin species tested, HcbA3 showed the highest affinity for FMN with a K d value of 0.75±0.17 µM. Kinetic assays revealed that HcbA3 followed a ping-pong bi-bi mechanism for the reduction of flavins. The K m for NADH and FMN was 51.66±11.58 µM and 4.43±0.69 µM, respectively. For both NADH and FMN, the V max and k cat were 2.21±0.86 µM and 66.74±5.91 sec-1, respectively. We also successfully reconstituted the oxidative dehalogenase reaction in vitro, which consisted of HcbA1, HcbA3, FMN, and NADH, suggesting that HcbA3 may be the partner reductase component for HcbA1 in Nocardioides sp. PD653.

Metabolite profiling reveals a complex response of plants to application of plant growth-promoting endophytic bacteria.

Endophytic bacteria have been explored for their role in plant growth promotion, however, not much has been explored in cucumber. The metabolomic response of plants to application of such microbes also remains largely unknown. Thus, we investigated the application of endophytic bacteria to cucumber to infer their role in plant growth promotion and document metabolome response. The lowest healthy leaf-stalks were sampled from four differently sourced cucumber plants, and endophytic bacteria were isolated after surface disinfection. Initial plant growth-promoting (PGP) screening was performed to identify PGP strains out of numerous isolates, and five strains (Strains 4=Curtobacterium spp., 72=Brevibacillus spp., 167=Paenibacillus spp., 193=Bacillus spp., and 227=Microbacterium spp.) were selected based on their contribution to root growth compared with the control. The selected strains were further evaluated in pot experiments, axenic PGP trait assays, and metabolomic analysis. Results revealed that the selected isolates possessed different qualitative characteristics among indole acetic acid, siderophore production, phosphate solubilization, and 1-aminocyclopropane-1-carboxylate (ACC)-deaminase and nifH genes, and all isolates significantly enhanced plant growth in both pot experiments compared with the uninoculated control and fertilizer control. Metabolomic profiling revealed that both strains affected the plant metabolomes compared with the uninoculated control. Around 50 % of the metabolites explored had higher concentrations in either or both bacteria-applied plants compared with the uninoculated control. Differences were observed in both strains' regulation of metabolites, although both enhanced root growth near equally. Overall, endophytic bacteria significantly enhanced plant growth and tended to produce or induce release of certain metabolites within the plant endosphere.

Dissipation, dehalogenation, and denitration of chloroaromatic compounds by Nocardioides sp. strain PD653: Characterization of the substrate specificity.

The substrate range of Nocardioides sp. strain PD653, capable of mineralizing hexachlorobenzene, was investigated based on the dissipation of substrates and the liberation of halogen ions. Strain PD653 dehalogenated 10 out of 18 halophenol congeners; however, it could dehalogenate only hexachlorobenzene out of seven halobenzene congeners tested. Moreover, dehalogenation activities were shown for chloronitrobenzenes, along with an increase in the number of substituted chlorine atoms except for 2,3,4,5-tetrachloro-1-nitrobenzene. These results suggested that this strain might be applicable to remediate soil contaminated with these persistent chloroaromatic compounds.

Multifunctional food waste fertilizer having the capability of Fusarium-growth inhibition and phosphate solubility: A new horizon of food waste recycle using microorganisms.

Organic waste, including food leftovers and trade refuse, has been explored for its use as a nutrient source through a multitude of techniques. Composting; the dominant method, is criticized due to exhaustion of nutrients used for simultaneous microbial growth. Drying of food waste to low moisture levels, besides keeping the nutrition intact, offers the potential of growing desirable phyto-beneficial-cum-functional microbes, which can have additional benefits. Consequently, isolation of fungus from soil was carried out followed by characterization for confrontation against Fusarium, phosphate solubilization and utilization of food waste material. The food waste material was collected from University of Yamanashi Restaurant and dried up to approximately 3.8% moisture using Hitachi Household Garbage Dryer & Processor (ECO-B25). A pot experiment, growing Lactuca sativa (lettuce) and Brassica rapa, in selected fungal isolate-inoculated food waste material was conducted comparing with that of chemical, and organic fertilizer besides uninoculated food waste material. Results showed that one strain UY2015_11 (identified as Aspergillus niger) significantly inhibited the growth of Fusarium besides solubilizing hardly available iron, and calcium-type phosphates. Similarly, in a 13-week incubation experiment, mineralization of nitrate nitrogen from the food waste and fungal strain UY2015_11-inoculated food waste, was 23.9% and 17.0%, respectively. Later pot experiment indicated that the strain UY2015_11-inoculated dried food waste material showed same vegetable growth as chemical and organic fertilizer (rapeseed oil cake). Concluding, Aspergillus niger strain UY2015_11 isolated from soil inhibited the growth of Fusarium and solubilized hardly phosphate. Moreover, the strain UY2015_11 inoculated low moisture-food waste material showed the same vegetable growth as chemical and organic fertilizer (rapeseed oil cake).

Changes in endophytic bacterial communities during different growth stages of cucumber (Cucumis sativus L.).

Endophytic bacterial diversity in plants presents the level of interaction between culturable and non-culturable endophytic bacteria, thereby providing an appropriate insight into the endophytic environment. This study was conducted to determine the trend of culturable and non-culturable endophytic bacteria at two different sites encompassing four consecutive growth stages. For culturable endophytic bacteria, isolation was carried out using the dilution plate technique, and the obtained colonies were compared using PCR-restriction fragment length polymorphism (RFLP). Different RFLP-types were identified to their nearest neighbour using 16S rRNA sequencing. The non-culturable endophytic bacterial diversity was obtained by next generation sequencing. Results suggested a similar trend among the culturable and non-culturable bacteria for observed operational taxonomic units and diversity indices. It is noticeable that the endophytic bacteria inhabiting in stage 1 disappeared, and instead, different endophytic bacteria appeared. Moreover, the temporal persistence of certain culturable and non-culturable bacteria was also observed. In conclusion, the endophytic bacterial diversity in cucumber initially increased with the plant growth and then decreased at a later stage. Furthermore, it was suggested that plants regulate the number and diversity of endophytes throughout the lifecycle of plants.

Potential use of endophytic root bacteria and host plants to degrade hydrocarbons.

Microbe-assisted phytoremediation depends on competent root-associated microorganisms that enhance remediation efficiency of organic compounds. Endophytic bacteria are a key element of the root microbiome and may assist plant degradation of contaminants. The aim of this study was to investigate the application of four hydrocarbon-degrading endophytic strains previously isolated from an oil sands reclamation area. Strains EA1-17 (Stenotrophomonas sp.), EA2-30 (Flavobacterium sp.), EA4-40 (Pantoea sp.), and EA6-5 (Pseudomonas sp.) were inoculated in white sweet clover growing on soils amended with diesel at 5,000, 10,000, and 20,000 mg·kg-1. Our results indicate that plant growth inhibition caused by diesel fuel toxicity was overcome in inoculated plants, which showed significantly higher plant biomass. Analysis of soil F2 and F3 hydrocarbon fractions also revealed that these soils were remediated by inoculated plants when diesel was applied at 10,000 mg·kg-1 and 20,000 mg·kg-1. In addition, quantification of hydrocarbon-degrading genes suggests that all bacterial strains successfully colonized sweet clover plants. Overall, the endophytic strain EA6-5 (Pseudomonas sp.), which harbored hydrocarbon-degrading genes, was the most effective candidate in phytoremediation experiments and could be a strategy to increase plant tolerance and hydrocarbon degradation in contaminated (e.g., diesel fuel) soils.

Biodegradability and biodegradation pathways of chlorinated cyclodiene insecticides by soil fungi.

An aerobic dieldrin-degrading fungus, Mucor racemosus strain DDF, and two aerobic endosulfan-degrading fungal strains, Mortierella sp. strains W8 and Cm1-45, were isolated from soil contaminated with organochlorine pesticides. Strain DDF degraded more than 90% dieldrin during 10-days of incubation at 25°C and showed the production of a small amount of aldrin trans-diol. Moreover, strain DDF reduced levels of aldrin trans-diol while producing unknown metabolites that were determined to be aldrin trans-diol exo- and endo-phosphates. On the other hand, Mortierella sp. strains W8 and Cm1-45 degraded more than 70% and 50% of α and ß-endosulfan, respectively, over 28 days at 25°C, in liquid cultures containing initial concentrations of 8.2 µM of each substance. Only a small amount of endosulfan sulfate, a persistent metabolite, was detected in the both cultures, while these strains could not degrade endosulfan sulfate when this compound was provided as the initial substrate. Both strains generate endosulfan diol as a first step in the degradation of endosulfan, then undergo further conversion to endosulfan lactone.

Isolation of endosulfan sulfate-degrading Rhodococcus koreensis strain S1-1 from endosulfan contaminated soil and identification of a novel metabolite, endosulfan diol monosulfate.

An aerobic endosulfan sulfate-degrading bacterium, Rhodococcus koreensis strain S1-1, was isolated from soil to which endosulfan had been applied annually for more than 10 years until 2008. The strain isolated in this work reduced the concentration of endosulfan sulfate (2) from 12.25 µM to 2.11 µM during 14 d at 30 °C. Using ultra performance liquid chromatography-electrospray ionization-mass spectroscopy (UPLC-ESI-MS), a new highly water-soluble metabolite possessing six chlorine atoms was found to be endosulfan diol monosulfate (6), derived from 2 by hydrolysis of the cyclic sulfate ester ring. The structure of 6 was elucidated by chemical synthesis of the candidate derivatives and by HR-MS and UPLC-MS analyses. Therefore, it was suggested that the strain S1-1 has a new metabolic pathway of 2. In addition, 6 was expected to be less toxic among the metabolites of 1 because of its higher water-solubility.

Arsenic biotransformation by Streptomyces†sp. isolated from rice rhizosphere.

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As-contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S-adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.

Biodegradability and biodegradation pathways of endosulfan and endosulfan sulfate.

Endosulfan and endosulfan sulfate are persistent organic pollutants that cause serious environmental problems. Although these compounds are already prohibited in many countries, residues can be detected in soils with a history of endosulfan application. Endosulfan is transformed in the environment into endosulfan sulfate, which is a toxic and persistent metabolite. However, some microorganisms can degrade endosulfan without producing endosulfan sulfate, and some can degrade endosulfan sulfate. Therefore, biodegradation has the potential to clean up soil contaminated with endosulfan. In this review, we provide an overview of aerobic endosulfan degradation by bacteria and fungi, and a summary of recent advances and prospects in this research field.

Detecting nonculturable bacteria in the active mycorrhizal zone of the pine mushroom Tricholoma matsutake.

The fungus Tricholoma matsutake forms an ectomycorrhizal relationship with pine trees. Its sporocarps often develop in a circle, which is commonly known as a fairy ring. The fungus produces a solid, compact, white aggregate of mycelia and mycorrhizae beneath the fairy ring, which in Japanese is called a 'shiro'. In the present study, we used soil dilution plating and molecular techniques to analyze the bacterial communities within, beneath, and outside the T. matsutake fairy ring. Soil dilution plating confirmed previous reports that bacteria and actinomycetes are seldom present in the soil of the active mycorrhizal zone of the T. matsutake shiro. In addition, the results showed that the absence of bacteria was strongly correlated with the presence of T. matsutake mycorrhizae. The results demonstrate that bacteria, especially aerobic and heterotrophic forms, and actinomycetes, are strongly inhibited by T. matsutake. Indeed, neither bacteria nor actinomycetes were detected in 11.3% of 213 soil samples from the entire shiro area by culture-dependent methods. However, molecular techniques demonstrated that some bacteria, such as individual genera of Sphingomonas and Acidobacterium, were present in the active mycorrhizal zone, even though they were not detected in soil assays using the dilution plating technique.

Biodegradation of endosulfan by Mortieralla sp. strain W8 in soil: Influence of different substrates on biodegradation.

To examine the bioremediation potential of Mortierella sp. strain W8 in endosulfan contaminated soil, the fungus was inoculated into sterilized and unsterilized soil spiked with endosulfan. Wheat bran and cane molasses were used as substrates to understand the influence of different organic materials on the degradation of endosulfan in soil. Strain W8 degraded α- and ß-endosulfan in both sterilized and unsterilized soil. In unsterilized soil with wheat bran+W8, α- and ß- endosulfan were degraded by approximately 80% and 50%, respectively after 28 d incubation against the initial endosulfan concentration (3 mg kg(-1) dw). The corresponding values for α- and ß-endosulfan degradation with wheat bran only were 50% and 3%. Endosulfan diol metabolite was detected after 14 d incubation in wheat bran+W8 whereas it was not found with wheat bran only. Production of endosulfan sulfate, the main metabolite of endosulfan, was suppressed with wheat bran+W8 treatment compared with wheat bran only. It was demonstrated that wheat bran is a more suitable substrate for strain W8 than cane molasses. Wheat bran+W8 is a superior fungus and substrate mix for bioremediation in soil contaminated with endosulfan.

Isolation and identification of dieldrin-degrading Pseudonocardia sp. strain KSF27 using a soil-charcoal perfusion method with aldrin trans-diol as a structural analog of dieldrin.

We isolated a novel aerobic dieldrin-degrading bacterium from an enrichment culture in a soil-charcoal perfusion system. Enrichment culture using a soil-charcoal perfusion system was an effective way to obtain microorganisms that degrade recalcitrant compounds. The soil-charcoal perfusion was performed using aldrin trans-diol, which was a metabolite of dieldrin. Aldrin trans-diol had higher bioavailability (2.5 mg/l) than dieldrin (0.1-0.25 mg/l), therefore it is possible for microorganisms to utilize it as a substrate in soil. After 100 days of circulation and three exchanges of the medium, the enriched charcoal was harvested and a bacterium isolated. The isolate was designated as strain KSF27 and was found to be closely related to Pseudonocardia spp. as determined by 16S rRNA sequencing analysis. Strain KSF27 degraded aldrin trans-diol by 0.05 µmol/l from an initial concentration of 25.5 µmol/l. The metabolite of aldrin trans-diol was detected by HPLC/MS and determined to be aldrindicarboxylic acid based on retention time and the MS fragment. Moreover, strain KSF27 degraded dieldrin from 14.06 µmol/l to 2.01 µmol/l over a 10-day incubation at 30°C. This strain degraded dieldrin and other persistent organochlorine pesticides, such as α-endosulfan, ß-endosulfan, endosulfan sulfate, heptachlor, heptachlor epoxide and chlordecone.