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Carbon nanosol (CNS) is a carbon-based nanomaterial capable of promoting plant growth while the underlying mechanism involved in this process remains unknown. This study demonstrates that CNS promotes rice seedling growth under restricted concentrations. Macroelement transporter mutants were investigated to further investigate the CNS-mediated promotion of rice seedling growth. The genetic and physiological findings revealed that nitrate transporter 1.1B (NRT1.1B) and ammonium transporter 1 (AMT1) mutants inhibited the CNS-induced growth development of rice seedlings, whereas potassium transporter (AKT1) and phosphate transporter 8 (PT8) did not exhibit any inhibitory effects. Further investigations demonstrated the inhibition of CNS-mediated growth promotion via glutamine synthetase 1;1 (gs1;1) mutants. Additionally, the administration of CNS resulted in enhanced accumulation of chlorophyll in plants, and the promotion of CNS-induced growth was inhibited by yellow-green leaf 8 (YGL8) mutants and the chlorophyll biosynthetic gene divinyl reductase (DVR) mutants. According to these findings, the CNS promotes plant growth by stimulating chlorophyll biosynthesis. Furthermore, the presence of CNS enhanced the ability of rice to withstand blast, sheath blight (ShB), and bacterial blight. The nrt1.1b, amt1, dvr, and ygl8 mutants did not exhibit a broad spectrum effect. The positive regulation of broad-spectrum resistance in rice by GS1;1 suggests the requirement of N assimilation for CNS-mediated broad-spectrum resistance. In addition, an in vitro assay demonstrated that CNS inhibits the growth of pathogens responsible for blast, ShB, and bacterial blight, namely Magnaporthe oryzae, Rhizoctonia solani AG1-IA, and Xanthomonas oryzae pv. Oryzae, respectively. CNS application may also induce broad-spectrum resistance against bacterial and fungal pathogens, indicating that in addition to its antifungal and antibacterial properties, CNS application may also stimulate N assimilation. Collectively, the results indicate that CNS may be a potential nano-therapeutic agent for improved plant growth promotion while also providing broad-spectrum resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: WD40 proteins, which are highly prevalent in eukaryotes, play important roles in plant development and stress responses. However, systematic identification and exploration of WD40 proteins in tobacco have not yet been conducted., Results: In this study, a total of 399 WD40 regulatory genes were identified in common tobacco (Nicotiana tabacum). Gene structure and motif analysis revealed structural and functional diversity among different clades of tobacco WD40 regulatory genes. The expansion of tobacco WD40 regulatory genes was mainly driven by segmental duplication and purifying selection. A potential regulatory network of NtWD40s suggested that NtWD40s might be regulated by miRNAs and transcription factors in various biological processes. Expression pattern analysis via transcriptome analysis and qRT-PCR revealed that many NtWD40s exhibited tissue-specific expression patterns and might be involved in various biotic and abiotic stresses. Furthermore, we have validated the critical role of NtTTG1, which was located in the nuclei of trichome cells, in enhancing the drought tolerance of tobacco plants., Conclusions: Our study provides comprehensive information to better understand the evolution of WD40 regulatory genes and their roles in different stress responses in tobacco., (© 2024. The Author(s).)

Tobacco plants (Nicotiana tabacum L.) exhibit considerable potential for phytoremediation of soil cadmium (Cd) pollutants, owing to their substantial biomass and efficient metal accumulation capabilities. The reduction of Cd accumulation in tobacco holds promise for minimizing Cd intake in individuals exposed to cigar smoking. NRAMP transporters are pivotal in the processes of Cd accumulation and resistance in plants; however, limited research has explored the functions of NRAMPs in tobacco plants. In this investigation, we focused on NtNRAMP6c, one of the three homologs of NRAMP6 in tobacco. We observed a robust induction of NtNRAMP6c expression in response to both Cd toxicity and iron (Fe) deficiency, with the highest expression levels detected in the roots. Subsequent subcellular localization and heterologous expression analyses disclosed that NtNRAMP6c functions as a plasma membrane-localized Cd transporter. Moreover, its overexpression significantly heightened the sensitivity of yeast cells to Cd toxicity. Through CRISPR-Cas9-mediated knockout of NtNRAMP6c, we achieved a reduction in Cd accumulation and an enhancement in Cd resistance in tobacco plants. Comparative transcriptomic analysis unveiled substantial alterations in the transcriptional profiles of genes associated with metal ion transport, photosynthesis, and macromolecule catabolism upon NtNRAMP6c knockout. Furthermore, our study employed plant metabolomics and rhizosphere metagenomics to demonstrate that NtNRAMP6c knockout led to changes in phytohormone homeostasis, as well as shifts in the composition and abundance of microbial communities. These findings bear significant biological implications for the utilization of tobacco in phytoremediation strategies targeting Cd pollutants in contaminated soils, and concurrently, in mitigating Cd accumulation in tobacco production destined for cigar consumption., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Carbon nanosol (CNS) is a carbon-based nanomaterial that promotes plant growth; however, its functional mechanisms and effects on the microbiome are not fully understood. Here, we explored the effects of CNS on the relationship between the soil, endophytic microbiomes and plant productivity. CNS treatment increased the fresh biomass of tobacco (Nicotiana tabacum L.) plants by 27.4% ± 9.9%. Amplicon sequencing analysis showed that the CNS treatment significantly affected the composition and diversity of the microbial communities in multiple ecological niches associated with tobacco, especially the bulk soil and stem endophytic microbiome. Furthermore, the application of CNS resulted in enhanced network connectivity and stability of the microbial communities in different niches, particularly in the soil, implying a strengthening of certain microbial interactions. Certain potentially growth-promoting root endophytic bacteria were more abundant under the CNS treatment. In addition, CNS increased the abundance of some endophytic microbial functional genes known to enhance plant growth, such as those associated with nutrient metabolism and the plant hormone biosynthesis pathways. We isolated two bacterial strains (Sphingopyxis sp. and Novosphingobium sp.) that were enriched under CNS treatment, and they were confirmed to promote tobacco plant growth in vitro. These results suggested that CNS might, at least in part, promote plant growth by enriching beneficial bacteria in the microbiome., (© 2023. The Author(s).)

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with low or no protein-coding ability, which play essential roles in various biological processes in plants. Tobacco is an ideal model plant for studying nicotine biosynthesis and metabolism, and there is little research on lncRNAs in this field. Therefore, how to take advantage of the mature tobacco system to profoundly investigate the lncRNAs involved in the nicotine pathway is intriguing. By exploiting 549 public RNA-Seq datasets of tobacco, 30,212 lncRNA candidates were identified, including 24,084 large intervening non-coding RNAs (lincRNAs), 5,778 natural antisense transcripts (NATs) and 350 intronic non-coding RNAs (incRNAs). Compared with protein-coding genes, lncRNAs have distinct properties in terms of exon number, sequence length, A/U content, and tissue-specific expression pattern. lincRNAs showed an asymmetric evolutionary pattern, with a higher proportion (68.71%) expressed from the Nicotiana sylvestris (S) subgenome. We predicted the potential cis/trans-regulatory effects on protein-coding genes. One hundred four lncRNAs were detected as precursors of 30 known microRNA (miRNA) family members, and 110 lncRNAs were expected to be the potential endogenous target mimics for 39 miRNAs. By combining the results of weighted gene co-expression network analysis with the differentially expressed gene analysis of topping RNA-seq data, we constructed a sub-network containing eight lncRNAs and 25 nicotine-related coding genes. We confirmed that the expression of seven lncRNAs could be affected by MeJA treatment and may be controlled by the transcription factor NtMYC2 using a quantitative PCR assay and gene editing. The results suggested that lncRNAs are involved in the nicotine pathway. Our findings further deepened the understanding of the features and functions of lncRNAs and provided new candidates for regulating nicotine biosynthesis in tobacco., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xie, Jin, Wang, Lu, Li, Tao, Cao and Xu.)

Root-knot nematode (RKN) disease is a destructive soil disease that affects crop health and causes huge losses in crop production. To explore the relationships between soil environments, rhizobacterial communities, and plant health, rhizosphere bacterial communities were analyzed using metagenomic sequencing in tobacco samples with different grades of RKN disease. The results showed that the community structure and function of the plant rhizosphere were significantly correlated to the RKN disease. RKN density and urease content were key factors affecting the rhizosphere bacterial community. Urease accelerated the catabolism of urea and led to the production of high concentrations of ammonia, which directly suppressed the development of RKNs or by improving the nutritional and growth status of microorganisms that were antagonistic to RKNs. Further experiments showed that the suppression role of ammonia should be attributed to the direct inhibition of NH 3 . The bacterial members that were positively correlated with RKN density, contained many plant cell wall degrading enzymes, which might destroy plant cell walls and promote the colonization of RKN in tobacco roots. The analysis of metatranscriptome and metabolism demonstrated the role of these cell wall degrading enzymes. This study offers a comprehensive insight into the relationships between RKNs, bacteria, and soil environmental factors and provides new ideas for the biological control of RKNs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microbial degradation of organic compounds is an environmentally benign and energy efficient part in product processing. Fermentation of plant leaves involves enzymatic actions of many microorganisms. However, microbes and enzymes discovered from natural degradation communities were still limited by cultural methods. In this study, we used a metagenomics sequence-guided strategy to identify the microbes and enzymes involved in compound degradation and explore the potential synergy among community members in fermented tobacco leaves. The results showed that contents of protein, starch, pectin, lignin, and cellulose varied in fermented leaves from different growing sites. The different compound contents were closely related to taxonomic composition and functional profiles of foliar microbial communities. Microbial communities showed significant correlations with protein, lignin, and cellulose. Vital species for degradations of protein (Bacillus cereus and Terribacillus aidingensis), lignin (Klebsiella pneumoniae and Pantoea ananatis) and cellulose (Pseudomonas putida and Sphingomonas sp. Leaf20) were identified and relating hydrolytic enzymes were annotated. Further, twenty-two metagenome-assembled genomes (MAGs) were assembled from metagenomes and six potential cellulolytic genomes were used to reconstruct the cellulose-degrading process, revealing the potential metabolic cooperation related to cellulose degradation. Our work should deepen the understanding of microbial roles in plant fermentation and provide a new viewpoint for applying microbial consortia to convert plant organic components to small molecules., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Long non-coding RNAs (lncRNAs) regulate many biological processes in plants, including defense against pathogens and herbivores. Recently, many small ORFs embedded in lncRNAs have been identified to encode biologically functional peptides (small ORF-encoded peptides [SEPs]) in many species. However, it is unknown whether lncRNAs mediate defense against herbivore attack and whether there are novel functional SEPs for these lncRNAs. By sequencing Spodoptera litura -treated leaves at six time-points in Nicotiana tabacum , 22,436 lncRNAs were identified, of which 787 were differentially expressed. Using a comprehensive mass spectrometry (MS) pipeline, 302 novel SEPs derived from 115 tobacco lncRNAs were identified. Moreover, 61 SEPs showed differential expression after S. litura attack. Importantly, several of these peptides were characterized through 3D structure prediction, subcellular localization validation by laser confocal microscopy, and western blotting. Subsequent bioinformatic analysis revealed some specific chemical and physical properties of these novel SEPs, which probably represent the largest number of SEPs identified in plants to date. Our study not only identifies potential lncRNA regulators of plant response to herbivore attack but also serves as a valuable resource for the functional characterization of SEP-encoding lncRNAs., Competing Interests: Authors KC, ZWL, and XY were employed by China Tobacco Hunan Industrial Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jin, Meng, Chen, Xu, Lu, Li, Tao, Li, Wang, Yang, Yu, Yang, Cao and Cao.)

Root-associated compartments, including the rhizosphere, rhizoplane, and endosphere, live with diverse microbial communities which profoundly affect plant growth and health. However, a systematic understanding of the microbiome assembly across the rhizosphere, rhizoplane, and endosphere under pathogen invasion remains elusive. Using 16S high-throughput sequencing, we studied how bacterial wilt disease affected the variation and assembly of the three continuous root-associated microbiomes of tobacco. The results indicated that microorganisms were gradually filtered from the rhizosphere to the endosphere. With the pathogen invasion, the rhizosphere, rhizoplane and endosphere microbiomes selected and recruited different beneficial bacterial taxa. Some recruited bacteria were also identified as keystone members in networks (i.e., Bosea in the endosphere). The microbiomes of endosphere and rhizoplane were more sensitive to plant disease than the rhizosphere microbiome. Still, response strategies of the rhizoplane and endosphere to disease were obviously different. Microbial networks of the rhizoplane became complex in diseased samples and genes involved in sporulation formation and cell cycle were enriched. However, microbial networks of the diseased endosphere were disrupted, and functional genes related to nitrogen utilization and chemotaxis were significantly increased, indicating the importance of nitrogen resources supply of plants for the endosphere microbiome under pathogen invasion. Our results provide novel insights for understanding the different responses of the root-associated microbiomes to plant disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tao, Yu, Jin, Lu, Yang, Xu, Chen, Li and Cao.)

The root phenotypic traits have been considered as important factors in shaping the rhizosphere microbiome and regulating plant growth. However, the relationships between root phenotypic traits and the rhizosphere bacterial community remain unclear. We investigated two fields with different developing tobacco roots by a long-term positioning test in Hengshi. The well-developed root system (WDR) showed much more superiority in root phenotypic traits, including total root length, total projection area, surface area, and root tip number, than the underdeveloped root system. The specific root traits in WDR provided more ecological niches for the rhizosphere microorganisms, contributing to a more diverse microbial community and a more complex microbial network. The total root length and root tip number were the key factors shaping bacterial communities in the rhizosphere. In turn, the phyla Acidobacteria and Bacteroidetes might play vital roles in modifying root development and promoting plant growth according to their positive correlation with root phenotypic traits. Linking root phenotypic traits to the microbiome may enhance our understanding of rhizospheric interactions and their roles in developing rhizosphere ecosystems., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The advent of single-cell sequencing opened a new era in transcriptomic and genomic research. To understand cell composition using single-cell studies, a variety of cell markers have been widely used to label individual cell types. However, the specific database of cell markers for use by the plant research community remains very limited. To overcome this problem, we developed the Plant Cell Marker DataBase (PCMDB, http://www.tobaccodb.org/pcmdb/), which is based on a uniform annotation pipeline. By manually curating over 130 000 research publications, we collected a total of 81 117 cell marker genes of 263 cell types in 22 tissues across six plant species. Tissue- and cell-specific expression patterns can be visualized using multiple tools: eFP Browser, Bar, and UMAP/TSNE graph. The PCMDB also supports several analysis tools, including SCSA and SingleR, which allows for user annotation of cell types. To provide information about plant species currently unsupported in PCMDB, potential marker genes for other plant species can be searched based on homology with the supported species. PCMDB is a user-friendly hierarchical platform that contains five built-in search engines. We believe PCMDB will constitute a useful resource for researchers working on cell type annotation and the prediction of the biological function of individual cells., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Continuous cropping has become the most common system in intensive, modern agricultural production; however, obstacles often appear in continuous cropping patterns after a few years of use. There have been several studies about the impacts of continuous cropping on soil microbial, but few about differences between soil experiencing continuous cropping obstacles and those where such obstacles had been resisted. Here, after ten or twenty years of continuous tobacco cropping, we collected soil samples investigating discrepancies in soil property and bacterial community between soils experiencing continuous cropping obstacles and soils where the obstacles were resisted providing insight into preventing and controlling continuous cropping obstacles. Results showed that soil organic matter (SOM), available phosphorus (AP), total nitrogen (TN), nitrate-N (NO 3 - -N), and bacterial diversity of samples where continuous cropping obstacles had been resisted were significantly higher than those where continuous cropping obstacles were present. Besides, SOM, AP, TN, and Ammonium-N (NH 4 + -N) considerably affected the bacterial community. Among all variables, NH 4 + -N explained the largest proportion of bacterial community variation. Molecular ecological networks were used to putatively identify keystone taxa, including Acidobacteria Gp1, Acidobacteria Gp2, Acidobacteria Gp16, and WPS-1_genera_incertae_sedis. Their relative abundance significantly changed between the two conditions. Overall, our results indicate that decreases in soil nutrient content and bacterial diversity, and significant changes in some keystone taxa abundances may be important factors leading to increased soil-borne diseases and reduced tobacco production potential or quality. Thus, during agricultural production, we could regulate the stability of the soil-crop-microbial ecological system via crop rotation, intercropping, or the use of specialized bio-fertilizers and soil conditioners to mitigate continuous cropping obstacles., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Plant leaves are colonized by a remarkably diverse fungal microbiome, which contributes to host plant growth and health. However, responses of foliar fungal community to phytopathogen invasion and measures of the fungal community taken to resist or assist pathogens remain elusive. By utilizing high-throughput sequencing of internal transcribed spacer (ITS) amplicons, we studied the relationships between the foliar fungal community around the disease spot and the pathogen of brown spot disease. The pathogenic Alternaria was found to follow a dramatically decreased trend from the disease spot to its surrounding fungal communities, whose community structure also diverged substantially away from the disease spot community. With the increase of pathogenic Alternaria, diversity indexes, including Shannon, Pielou and Simpson, showed a trend of increasing first and then decreasing. Total network links and the average path distance exhibited strong negative and positive correlations with Alternaria, respectively. Five keystone members showed direct interactions with pathogenic Alternaria. Members of Botryosphaeria, Paraphoma and Plectosphaerella might act as key 'pathogen facilitators' to increase the severity and development of brown spot disease, while Pleospora and Ochrocladosporium might be important 'pathogen antagonists' to suppress the expansion of pathogenic Alternaria. Our study provides new insights in developing new strategies for leaf disease prediction or prevention., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alicyclobacillus species inhabit diverse environments and have adapted to broad ranges of pH and temperature. However, their adaptive evolutions remain elusive, especially regarding the role of mobile genetic elements (MGEs). Here, we characterized the distributions and functions of MGEs in Alicyclobacillus species across five environments, including acid mine drainage (AMD), beverages, hot springs, sediments, and soils. Nine Alicyclobacillus strains were isolated from AMD and possessed larger genome sizes and more genes than those from other environments. Four AMD strains evolved to be mixotrophic and fell into distinctive clusters in phylogenetic tree. Four types of MGEs including genomic island (GI), insertion sequence (IS), prophage, and integrative and conjugative element (ICE) were widely distributed in Alicyclobacillus species. Further, AMD strains did not possess CRISPR-Cas systems, but had more GI, IS, and ICE, as well as more MGE-associated genes involved in the oxidation of iron and sulfide and the resistance of heavy metal and low temperature. These findings highlight the differences in phenotypes and genotypes between strains isolated from AMD and other environments and the important role of MGEs in rapid environment niche expansions., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: To investigate the ecological effects of chemical and biological control methods on tobacco wildfire disease, a plot field experiment was conducted to compare the control efficiency and mechanisms of a chemical pesticide (kasugamycin wettable powder, KWP) and a biological control agent (BCA) through high-throughput sequencing of bacterial 16S rRNA genes., Results: The results showed that the BCA displayed better performance in decreasing the disease index and morbidity of tobacco than the chemical pesticide. By monitoring the endophytic community within tobacco leaves, it was found that the control effects of these two methods might be mediated by different changes in the endophytic bacterial communities and community assembly patterns. The application of either method decreased the taxonomic diversity of the leaf endophytic community. Compared to the BCA, KWP showed a more significant effect on the endophytic community structure, while the endophytic community treated with the BCA was able to return to the original state, which presented much lower disease infection. The disease control efficiency of KWP and BCA treatments might be achieved by increasing the abundance of Sphingomonas and Streptophyta, respectively. Furthermore, an analysis of the ecological processes in community assembly indicated that the BCA strengthened the homogeneous and variable selection, while KWP enhanced ecological drift., Conclusions: The results suggested different control mechanisms between KWP and BCA treatments, which will help in developing diverse ecological strategies for plant disease control.

Six artificial communities with different function or biodiversity were reconstructed by six typical bioleaching species for chalcopyrite leaching. Absence of sulfur oxidizers in communities significantly reduced copper extraction rates, and low diversity communities also exhibited slightly poor bioleaching performances. The variations of pH, redox potential, ferrous and copper ions indicated that the community with both sulfur oxidizers and high diversity showed fast adaptation to the environment and rapid dissolution of chalcopyrite. Integrated analysis of mineralogical and microbial parameters demonstrated that functional types of microorganisms made more contributions in mediating chalcopyrite dissolution than microbial diversity. Further correlation analysis between microbial types and chalcopyrite dissolution performances showed that sulfur oxidizers, especially Acidithiobacillus caldus, could greatly accelerate chalcopyrite dissolution by regulating solution physicochemical factors, such as redox potential and pH. This study provided a theoretical basis for improving bioleaching efficiency by balancing microbial functional types and biodiversity., (Copyright © 2020. Published by Elsevier Ltd.)

The interaction between microorganisms and minerals was a hot topic to reveal the transformation of key elements that affecting bioleaching efficiency. Three typical low-grade copper ores, the main copper-bearing components of which were primary sulfide, secondary sulfide and high-oxidative sulfide copper, were obtained from Dexing, Zijinshan and Luanshya copper mine, respectively. Meanwhile, six typical microorganisms were isolated from each of the three habitats, and assembled as communities based on their origins. Cross bioleaching was carried out under identical conditions. The leaching parameters showed that native strains played excellent roles in their corresponding ore bioleaching process, and community structure was greatly determined by mineral composition, indicating that domestication for longitudinal adaption was an effective way to improve microbial leaching performance. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans promoted copper release by shifting redox potential and pH of the leachate, respectively, indicating that microbial population regulation was another effective way to improve bioleaching efficiency., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The improper stacking of chromium (Cr) slag poses a great threat to the environment and human health. The toxicity of Cr in soil is not only related to its total amount, but also to its fractions. A simulated experiment was conducted in laboratory to assess the environmental risk of Cr fractions migration and distribution in red soil. The results showed the content of acid-soluble and reducible Cr significantly decreased ( P < 0.05) in top layer but increased in middle and substratum layers over time. This indicated that acid-soluble and reducible Cr migrated downward with time and the relative mobility of acid-soluble Cr (0.038 mg/kg·d·m) was higher than that of reducible Cr (0.028 mg/kg·d·m). Furthermore, correlation analysis between microbial community and chromium fraction showed the relative abundance of Lysobacter , Flavihumibacter , Flavisolbacter, and Altererythrobacter was significantly ( P < 0.05) correlated with acid-soluble and reducible fractions. Thus, these microorganisms might be evaluators to assess the migration of acid-soluble and reducible fractions in red soil. In summary, this study provided a new comprehension on remediation of Cr-contaminated soil by monitoring the migration of acid-soluble and reducible fractions and the changes of related microbial groups.

The phyllosphere supports a tremendous diversity of microbes, which have the potential to influence plant biogeography and ecosystem function. Although biocontrol agents (BCAs) have been used extensively for controlling plant diseases, the ecological effects of BCAs on phyllosphere bacteria and the relationships between phyllosphere community and plant health are poorly understood. In this study, we explored the control efficiency of two BCA communities on bacterial wildfire disease by repeatedly spraying BCAs on tobacco leaves. The results of field tests showed that BCAs used in our study, especially BCA&#95;B, had remarkable control effects against tobacco wildfire disease. The higher control efficiency of BCA&#95;B might be attributed to a highly diverse and complex community in the phyllosphere. By 16S ribosomal RNA gene sequencing, we found that phyllosphere microbial community, including community diversity, taxonomic composition and microbial interactions, changed significantly by application of BCAs. According to the correlation analysis, it showed that wildfire disease infection of plants was negatively related to phyllosphere microbial diversity, indicating a highly diverse community in the phyllosphere might prevent pathogens invasion and colonization. In addition, we inferred that a more complex network in the phyllosphere might be beneficial for decreasing the chances of bacterial wildfire outbreak, and the genera of Pantoea and Sphingomonas might play important roles in wildfire disease suppression. These correlations between phyllosphere community and plant health will improve our understanding on the ecological function of phyllosphere community on plants.

Members of the genus Acidithiobacillus , which can adapt to extremely high concentrations of heavy metals, are universally found at acid mine drainage (AMD) sites. Here, we performed a comparative genomic analysis of 37 strains within the genus Acidithiobacillus to answer the untouched questions as to the mechanisms and the evolutionary history of metal resistance genes in Acidithiobacillus spp. The results showed that the evolutionary history of metal resistance genes in Acidithiobacillus spp. involved a combination of gene gains and losses, horizontal gene transfer (HGT), and gene duplication. Phylogenetic analyses revealed that metal resistance genes in Acidithiobacillus spp. were acquired by early HGT events from species that shared habitats with Acidithiobacillus spp., such as Acidihalobacter , Thiobacillus , Acidiferrobacter , and Thiomonas species. Multicopper oxidase genes involved in copper detoxification were lost in iron-oxidizing Acidithiobacillus ferridurans , Acidithiobacillus ferrivorans , and Acidithiobacillus ferrooxidans and were replaced by rusticyanin genes during evolution. In addition, widespread purifying selection and the predicted high expression levels emphasized the indispensable roles of metal resistance genes in the ability of Acidithiobacillus spp. to adapt to harsh environments. Altogether, the results suggested that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. This study sheds light on the distribution, organization, functionality, and complex evolutionary history of metal resistance genes in Acidithiobacillus spp. IMPORTANCE Horizontal gene transfer (HGT), natural selection, and gene duplication are three main engines that drive the adaptive evolution of microbial genomes. Previous studies indicated that HGT was a main adaptive mechanism in acidophiles to cope with heavy-metal-rich environments. However, evidences of HGT in Acidithiobacillus species in response to challenging metal-rich environments and the mechanisms addressing how metal resistance genes originated and evolved in Acidithiobacillus are still lacking. The findings of this study revealed a fascinating phenomenon of putative cross-phylum HGT, suggesting that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. Altogether, the insights gained in this study have improved our understanding of the metal resistance strategies of Acidithiobacillus spp., (Copyright © 2019 American Society for Microbiology.)

Introducing exogenous species into an environment is an effective method to strengthen ecological functions. The traits of the exogenous species and the indigenous communities, as well as the resistance and subsequent succession of the community to exogenous species, are not well-understood. Here, three different functional consortia were introduced into two extremely acidic systems, leaching heap (LH) and leaching solution (LS), derived from the Zijin copper mine in China. The results showed that the structures of both LS and LH communities were affected by the three consortia, but not all the structural changes were in line with variations of community function. Among the three consortia, only the complementary sulfur oxidizers greatly enhanced copper extraction efficiency of LS (by 50.42%). This demonstrated that functional niche novelty gave exogenous species an advantage to occupy an ecological niche in a complementary manner, thus leading to successful colonization. The resistance to, and subsequent succession by, exogenous organisms varied between the two indigenous communities. More specifically, the LS community with low community diversity and simple composition was susceptible to exogenous species, and the community structural changes of LS were both divergent and irreversible. In comparison, the LH community with greater community diversity and more complex composition was more resistant to exogenous species, with the community structures showing a convergent trend over time despite different species being introduced. Therefore, we propose that diverse communities compete for resources more intensely with exogenous species and resist their introduction, and that communities with complex composition are able to cope with exogenous disturbances.

Introducing exogenous species to an indigenous microbial community is an effective way to reveal the connections between metabolic processes, ecological function and microbial community structure. Herein, three different functional consortia (ferrous oxidizers, sulfur oxidizers and ferrous/sulfur oxidizers) were added to a natural leaching solution system derived from Zijin copper mine, China. The leaching experiment showed that the copper extraction rate of the community invaded by a sulfur-oxidizing consortium was 50.40% higher than that of the indigenous leachate at the endpoint of bioleaching. The variations of ferrous content, total iron, pH and redox potential in leachates also provided evidence that the community with exogenous sulfur oxidizers was more efficient. XRD analysis demonstrated that a proper addition of the sulfur-oxidizing consortium could eliminate sulfur passivation, promote production of chalcocite and enhance leaching. Furthermore, an exogenous ferrous-oxidizing consortium and a sulfur-oxidizing consortium greatly changed the community structure and microbial succession and promoted the cell growth rate during the bioleaching process, while ferrous/sulfur oxidizers showed no obvious effects on the indigenous community. Exogenous ferrous oxidizers, mainly L. ferriphilum , and sulfur oxidizers, mainly A. thiooxidans , successfully established and colonized in the indigenous community. However, only colonized A. thiooxidans , rather than L. ferriphilum , showed advantageous enhancement in the dissolution of chalcopyrite. Results indicated that exogenous sulfur oxidizer A. thiooxidans , which was scarce in the indigenous community, could easily colonize in the indigenous community, significantly change the community structure, sufficiently execute its function, and greatly enhance copper dissolution., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Microbes play a critical role in soil global biogeochemical circulation and microbe-microbe interactions have also evoked enormous interests in recent years. Utilization of green manures can stimulate microbial activity and affect microbial composition and diversity. However, few studies focus on the microbial interactions or detect the key functional members in communities. With the advances of metagenomic technologies, network analysis has been used as a powerful tool to detect robust interactions between microbial members. Here, random matrix theory-based network analysis was used to investigate the microbial networks in response to four different green manure fertilization regimes (Vicia villosa, common vetch, milk vetch, and radish) over two growth cycles from October 2012 to September 2014. The results showed that the topological properties of microbial networks were dramatically altered by green manure fertilization. Microbial network under milk vetch amendment showed substantially more intense complexity and interactions than other fertilization systems, indicating that milk vetch provided a favorable condition for microbial interactions and niche sharing. The shift of microbial interactions could be attributed to the changes in some major soil traits and the interactions might be correlated to plant growth and production. With the stimuli of green manures, positive interactions predominated the network eventually and the network complexity was in consistency with maize productivity, which suggested that the complex soil microbial networks might benefit to plants rather than simple ones, because complex networks would hold strong the ability to cope with environment changes or suppress soil-borne pathogen infection on plants. In addition, network analyses discerned some putative keystone taxa and seven of them had directly positive interactions with maize yield, which suggested their important roles in maintaining environmental functions and in improving plant growth.

Bacterial wilt, caused by Ralstonia solanacearum, occurs occasionally during tobacco planting and potentially brings huge economic losses in affected areas. Soil microbes in different management stages play important roles in influencing bacterial wilt incidence. Studies have focused on the impacts of species diversity and composition during cropping periods on disease morbidity; however, the effects of the soil bacterial biomass, species diversity, species succession, and population interactions on morbidity remain unclear during non-cropping periods. In this study, we explored the soil bacterial communities in the non-cropping winter fallow (WF) and cropping late growing (LG) periods under consecutive monoculture systems using 16S ribosomal RNA gene sequencing and qPCR and further analyzed their effects on tobacco bacterial wilt incidence. We found that the bacterial communities in the WF period were significantly different from those in the LG period based on detrended correspondence analysis and dissimilarity tests. Crop morbidity was significantly related to bacterial community structure and to the presence of some genera during WF and LG periods. These genera, such as Arthrobacter, Pseudomonas, Acidobacteria GP6, and Pasteuria, may be potential biological control agents for bacterial wilt. Further analysis indicated that low soil bacterial diversity during the WF period, decrease of bacterial interactions from the WF to LG periods, and low soil biomass during the LG period all have the potential to increase morbidity. In conclusion, an increase of soil bacterial diversity and control of some bacterial abundances in the WF period might be an effective approach in controlling bacterial wilt incidence.

Acidithiobacillus ferrivorans is an acidophile that often occurs in low temperature acid mine drainage, e.g., that located at high altitude. Being able to inhabit the extreme environment, the bacterium must possess strategies to copy with the survival stress. Nonetheless, information on the strategies is in demand. Here, genomic and transcriptomic assays were performed to illuminate the adaptation mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine drainage environment in Yulong copper mine in southwest China. Genomic analysis revealed that strain has a gene repertoire for metal-resistance, e.g., genes coding for the mer operon and a variety of transporters/efflux proteins, and for low pH adaptation, such as genes for hopanoid-synthesis and the sodium:proton antiporter. Genes for various DNA repair enzymes and synthesis of UV-absorbing mycosporine-like amino acids precursor indicated hypothetical UV radiation-resistance mechanisms in strain YL15. In addition, it has two types of the acquired immune system-type III-B and type I-F CRISPR/Cas modules against invasion of foreign genetic elements. RNA-seq based analysis uncovered that strain YL15 uses a set of mechanisms to adapt to low temperature. Genes involved in protein synthesis, transmembrane transport, energy metabolism and chemotaxis showed increased levels of RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher RNA transcript counts at 6°C, possibly implicated in protecting DNA against oxidative stress at low temperature. The study represents the first to comprehensively unveil the adaptation mechanisms of an acidophilic bacterium to the acid mine drainage in alpine regions.

The use of green manures in agriculture can provide nutrients, affect soil microbial communities, and be a more sustainable management practice. The activities of soil microbes can effect crop growth, but the extent of this effect on yield remains unclear. We investigated soil bacterial communities and soil properties under four different green manure fertilization regimes (Vicia villosa, common vetch, milk vetch, and radish) and determined the effects of these regimes on maize growth. Milk vetch showed the greatest potential for improving crop productivity and increased maize yield by 31.3 %. This change might be related to changes in soil microbes and soil properties. The entire soil bacterial community and physicochemical properties differed significantly among treatments, and there were significant correlations between soil bacteria, soil properties, and maize yield. In particular, abundance of the phyla Acidobacteria and Verrucomicrobia was positively correlated with maize yield, while Proteobacteria and Chloroflexi were negatively correlated with yield. These data suggest that the variation of maize yield was related to differences in soil bacteria. The results also indicate that soil pH, alkali solution nitrogen, and available potassium were the key environmental factors shaping soil bacterial communities and determining maize yields. Both soil properties and soil microbes might be useful as indicators of soil quality and potential crop yield.

Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO4 was better than that cultured with the S(0) substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed.

Acidithiobacillus thiooxidans known for its ubiquity in diverse acidic and sulfur-bearing environments worldwide was used as the research subject in this study. To explore the genomic fluidity and intraspecific diversity of Acidithiobacillus thiooxidans (A. thiooxidans) species, comparative genomics based on nine draft genomes was performed. Phylogenomic scrutiny provided first insights into the multiple groupings of these strains, suggesting that genetic diversity might be potentially correlated with their geographic distribution as well as geochemical conditions. While these strains shared a large number of common genes, they displayed differences in gene content. Functional assignment indicated that the core genome was essential for microbial basic activities such as energy acquisition and uptake of nutrients, whereas the accessory genome was thought to be involved in niche adaptation. Comprehensive analysis of their predicted central metabolism revealed that few differences were observed among these strains. Further analyses showed evidences of relevance between environmental conditions and genomic diversification. Furthermore, a diverse pool of mobile genetic elements including insertion sequences and genomic islands in all A. thiooxidans strains probably demonstrated the frequent genetic flow (such as lateral gene transfer) in the extremely acidic environments. From another perspective, these elements might endow A. thiooxidans species with capacities to withstand the chemical constraints of their natural habitats. Taken together, our findings bring some valuable data to better understand the genomic diversity and econiche adaptation within A. thiooxidans strains.