Pseudomonas beijingensis sp. nov., a novel species widely colonizing plant rhizosphere.

A polyphasic taxonomic approach was used to characterize the three bacterial strains (FP830T, FP2034, and FP2262) isolated from the rhizosphere soil of rice, corn, and highland barley in Beijing, Heilongjiang, and Tibet, respectively, in PR China. These strains were Gram-negative, rod-shaped, and have one or two polar flagella. They exhibited optimal growth at 28 °C and pH 7.0 in the presence of 1 % (w/v) NaCl and showed fluorescence under ultraviolet light when cultivated on King's B plates. The FP830T genome size is 6.4 Mbp with a G+C content of 61.0 mol%. FP830T has the potential to promote plant growth by producing various metabolites such as fengycin, pyoverdin, indole-3-acetic acid, and the volatile substance 2,3-butanediol. Phylogenetic analysis indicated that three isolates formed an independent branch, which most closely related to type strains Pseudomonas thivervalensis DSM 13194T and Pseudomonas zanjanensis SWRI12T. The values of average nucleotide identity and digital DNA-DNA hybridization between three isolates and closest relatives were not higher than 93.7 and 52.3 %, respectively. The dominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and aminophospholipid. The predominant respiratory quinone was ubiquinone (Q-9). Based on polyphasic taxonomic analysis, it was concluded that strains FP830T, FP2034, and FP2262 represented a novel species within the genus Pseudomonas, and Pseudomonas beijingensis sp. nov. was proposed for the name of novel species. The type strain is FP830T (=ACCC 62448T=JCM 35689T).

Pseudomonas hefeiensis sp. nov., isolated from the rhizosphere of multiple cash crops in China.

Three bacterial strains, FP250T, FP821, and FP53, were isolated from the rhizosphere soil of oilseed rape, licorice, and habanero pepper in Anhui Province, Xinjiang Uygur Autonomous Region, and Jiangsu Province, PR China, respectively. All strains were shown to grow at 4-37 °C and pH 6.0-9.0, and in the presence of 0-4.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences or housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) and phylogenomic analysis showed that strains FP250T, FP821, and FP53 belong to the genus Pseudomonas, and are closely related to Pseudomonas kilonensis DSM 13647T, Pseudomonas brassicacearum JCM 11938T, Pseudomonas viciae 11K1T, and Pseudomonas thivervalensis DSM 13194T. The DNA G+C content of strain FP205T was 59.8 mol%. The average nucleotide identity and digital DNA-DNA hybridization values of strain FP205T with the most closely related strain were 93.2 % and 51.4 %, respectively, which is well below the threshold for species differentiation. Strain FP205T contained summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) as major fatty acids, and diphosphatidylglycerol along with phosphatidylethanolamine and aminophospholipid as major polar lipids. The predominant isoprenoid quinone was ubiquinone-9. Based on these phenotypic, phylogenetic, and chemotaxonomic results, strain FP205T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas hefeiensis sp. nov. is proposed. The type strain is FP205T (=ACCC 62447T=JCM 35687T).

Pseudomonas cucumis sp. nov., isolated from the rhizosphere of crop plants.

Two bacterial strains, FP1935T and FP1962, were isolated from the rhizosphere soil of cucumber and Chieh-qua plants, respectively, in Jilin Province, PR China. These strains were Gram-stain-negative, aerobic, rod-shaped and motile with one or two polar flagella. Analysis of the 16S rRNA gene sequences revealed that they represented members of the genus Pseudomonas, with the highest similarity to Pseudomonas silesiensis A3T (99.45 %), Pseudomonas frederiksbergensis JAJ28T (99.45 %), Pseudomonas mandelii NBRC 103147T (99.38 %), Pseudomonas piscium P50T (99.27 %) and Pseudomonas meliae CFBP 3225T (99.18 %). The DNA G+C contents of FP1935T and FP1962 were 58.99 mol% and 58.98 mol%, respectively. The results of in silico genome-based analyses indicated that these strains were distinct from other species in the genus Pseudomonas, as the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the recommended thresholds of 95 % (ANI) and 70 % (dDDH) for prokaryotic species delineation, with no values exceeding 94.1 and 55.8 %, respectively, compared with any other related species. The results of phenotypic and chemotaxonomic tests confirmed their differentiation from their closest relatives. The fatty acid profiles of both strains mainly consisted of summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C12 : 0 and C16 : 0. The predominant respiratory quinone was Q-9. Polar lipids include phosphatidylethanolamine, unidentified aminophospholipids, unidentified lipids and an unidentified phospholipid. On the basis of these phenotypic and genotypic results, we propose the name Pseudomonas cucumis sp. nov. for these novel strains. The type strain is FP1935T (=ACCC 62445T=JCM 35690T).

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

Pseudomonas syringae AlgU Downregulates Flagellin Gene Expression, Helping Evade Plant Immunity.

Flagella power bacterial movement through liquids and over surfaces to access or avoid certain environmental conditions, ultimately increasing a cell's probability of survival and reproduction. In some cases, flagella and chemotaxis are key virulence factors enabling pathogens to gain entry and attach to suitable host tissues. However, flagella are not always beneficial; both plant and animal immune systems have evolved receptors to sense the proteins that make up flagellar filaments as signatures of bacterial infection. Microbes poorly adapted to avoid or counteract these immune functions are unlikely to be successful in host environments, and this selective pressure has driven the evolution of diverse and often redundant pathogen compensatory mechanisms. We tested the role of AlgU, the Pseudomonas extracytoplasmic function sigma factor σE/σ22 ortholog, in regulating flagellar expression in the context of Pseudomonas syringae-plant interactions. We found that AlgU is necessary for downregulating bacterial flagellin expression in planta and that this results in a corresponding reduction in plant immune elicitation. This AlgU-dependent regulation of flagellin gene expression is beneficial to bacterial growth in the course of plant infection, and eliminating the plant's ability to detect flagellin makes this AlgU-dependent function irrelevant for bacteria growing in the apoplast. Together, these results add support to an emerging model in which P. syringae AlgU functions at a key control point that serves to optimize the expression of bacterial functions during host interactions, including minimizing the expression of immune elicitors and concomitantly upregulating beneficial virulence functions.IMPORTANCE Foliar plant pathogens, like Pseudomonas syringae, adjust their physiology and behavior to facilitate host colonization and disease, but the full extent of these adaptations is not known. Plant immune systems are triggered by bacterial molecules, such as the proteins that make up flagellar filaments. In this study, we found that during plant infection, AlgU, a gene expression regulator that is responsive to external stimuli, downregulates expression of fliC, which encodes the flagellin protein, a strong elicitor of plant immune systems. This change in gene expression and resultant change in behavior correlate with reduced plant immune activation and improved P. syringae plant colonization. The results of this study demonstrate the proximate and ultimate causes of flagellar regulation in a plant-pathogen interaction.

Genome-wide investigation of the genes involved in nicotine metabolism in Pseudomonas putida J5 by Tn5 transposon mutagenesis.

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain isolated from the tobacco rhizosphere. We successfully performed a comprehensive whole-genome analysis of nicotine metabolism-associated genes by Tn5 transposon mutagenesis in P. putida J5. A total of 18 mutants with unique insertions screened from 16,324 Tn5-transformants failed to use nicotine as the sole carbon source. Flanking sequences of the Tn5 transposon were cloned with a shotgun method from all of the nicotine-growth-deficient mutants. The potentially essential products of mutated gene were classified as follows: oxidoreductases, protein and metal transport systems, proteases and peptidases, transcriptional and translational regulators, and unknown proteins. Bioinformatic analysis of the Tn5 insertion sites indicated that the nicotine metabolic genes were separated and widely distributed in the genome. One of the mutants, M2022, was a Tn5 insert into a gene encoding a homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in Arthrobacter nicotinovorans. Genetic and biochemical analysis confirmed that three open reading frames (ORFs) from an approximately 13-kb fragment recovered from the mutant M2022 were responsible for the transformation of nicotine to 3-succinoyl-pyridine via pseudooxynicotine and 3-succinoyl semialdehyde-pyridine, the first three steps of nicotine degradation. Further research on these mutants and the Tn5-inserted genes will help us characterize nicotine metabolic processes in P. putida J5.

Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana.

Bacterial flagellin is perceived as a microbe (or pathogen)-associated molecular pattern (MAMP or PAMP) by the extracellular pattern recognition receptors, FLS2 and TLR5, of plants and mammals respectively. Flagellin accidently translocated into mammalian cells by pathogen type III secretion systems (T3SSs) is recognized by nucleotide-binding leucine-rich repeat receptor NLRC4 as a pattern of pathogenesis and induces a death-associated immune response. The non-pathogen Pseudomonas fluorescens Pf0-1, expressing a Pseudomonas syringae T3SS, and the plant pathogen P. syringae pv. tomato DC3000 were used to seek evidence of an analogous cytoplasmic recognition system for flagellin in the model plant Nicotiana benthamiana. Flagellin (FliC) was secreted in culture and translocated into plant cells by the T3SS expressed in Pf0-1 and DC3000 and in their ΔflgGHI flagellar pathway mutants. ΔfliC and ΔflgGHI mutants of Pf0-1 and DC3000 were strongly reduced in elicitation of reactive oxygen species production and in immunity induction as indicated by the ability of challenge bacteria inoculated 6 h later to translocate a type III effector-reporter and to elicit effector-triggered cell death. Agrobacterium-mediated transient expression in N. benthamiana of FliC with or without a eukaryotic export signal peptide, coupled with virus-induced gene silencing of FLS2, revealed no immune response that was not FLS2 dependent. Transiently expressed FliC from DC3000 and Pectobacterium carotovorum did notinduce cell death in N. benthamiana, tobacco or tomato leaves. Flagellin is the major Pseudomonas MAMP perceived by N. benthamiana, and although flagellin secretion through the plant cell wall by the T3SS may partially contribute to FLS2-dependent immunity, flagellin in the cytosol does not elicit immune-associated cell death. We postulate that a death response to translocated MAMPs would produce vulnerability to the many necrotrophic pathogens of plants, such as P. carotovorum, which differ from P. syringae and other (hemi)biotrophic pathogens in benefitting from death-associated immune responses.

Phenotypic and Genomic Characterization of Pseudomonas wuhanensis sp. nov., a Novel Species with Promising Features as a Potential Plant Growth-Promoting and Biocontrol Agent.

Plant growth-promoting rhizobacterial strain FP607T was isolated from the rhizosphere of beets in Wuhan, China. Strain FP607T exhibited significant antagonism toward several phytopathogenic bacteria, indicating that FP607T may produce antimicrobial metabolites and has a stronger biocontrol efficacy against plant pathogens. Growth-promoting tests showed that FP607T produced indole-3-acetic acid (IAA), NH3, and ferritin. The genome sequence of strain FP607T was 6,590,972 bp long with 59.0% G + C content. The optimum temperature range was 25-30 °C, and the optimum pH was 7. The cells of strain FP607T were Gram-negative, short, and rod-shaped, with polar flagella. The colonies on the King's B (KB) agar plates were light yellow, smooth, and circular, with regular edges. A phylogenetic analysis of the 16S rRNA sequence and a multilocus sequence analysis (MLSA) showed that strain FP607T was most closely related to the type of strain Pseudomonas farris SWRI79T. Based on a polyphasic taxonomic approach, strain FP607T was identified as a novel species within the genus Pseudomonas, for which the name Pseudomonas wuhanensis sp. nov. was proposed. The type of strain used was FP607T (JCM 35688, CGMCC 27743, and ACCC 62446).

Effects of the invasion of Ralstonia solanacearum on soil microbial community structure in Wuhan, China.

This study investigated the change in the microbiome of tomato rhizosphere soils after the invasion of Ralstonia solanacearum and analyzed the correlation between microbes and soil physicochemical properties. Diversity analyses of the bacteria in healthy and diseased rhizosphere soil samples (HRS and DRS) revealed that HRS had a higher species diversity and were compositionally different from DRS (P ≤ 0.05). Substantial differences in the relative abundance of Actinobacteria (37.52% vs 28.96%, P ≤ 0.05) and Proteobacteria (29.20% vs 35.59%, P ≤ 0.05) were identified in HRS and DRS, respectively. Taxonomic composition analysis showed ten differentially abundant genera, and seven of them (Gaiella, Roseisolibacter, Solirubrobacter, Kribbella, Acidibacter, Actinomarinicola, and Marmoricola) are more abundant in HRS. Soil pH and enzyme activities were negatively correlated with the abundance of R. solanacearum. The contents of total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkaline nitrogen (alkaline N), available phosphorus (AP), available potassium (AK), NO3-N(NN), NH4+-N (AN), and organic matter (OM) were all significantly increased in DRS. The composition and richness of protozoa in the samples show significant differences. Cephalobus, Acrobeles, Heteromita, norank_Tylenchida, and Rotylenchulus were enriched in DRS. Microbial interaction networks revealed that the HRS networks were more complex than the DRS networks. Overall, the results of this study demonstrate that healthy soil has a more complex microbial community structure and higher enzyme activity, and the invasion of R. solanacearum damages the soil microbial system.IMPORTANCEHow does the invasion of Ralstonia solanacearum affect tomato rhizosphere bacteria and protozoa? Which microbial changes can affect the growth of R. solanacearum? To date, most research studies focus on bacteria, with little research on protozoa, and even less on the synergistic effects between protozoa and bacteria. Here, we analyzed the correlation between tomato rhizosphere bacterial and protozoan communities and soil physicochemical properties during the invasion of R. solanacearum. We found that the diversity and abundance of rhizosphere microorganisms in healthy rhizosphere soil samples (HRS) were significantly higher than those in diseased rhizosphere soil samples (DRS), and there were significant changes in soil pH and enzyme activity. Overall, in this study, the analysis of microbial changes during the invasion of R. solanacearum provides a theoretical basis for the prevention and control of bacterial wilt.

Multiple lessons from the multiple functions of a regulator of type III secretion system assembly in the plant pathogen Pseudomonas syringae.

The assembly of type III secretion systems (T3SSs), which inject bacterial effector proteins into the cytosol of animal and plant hosts, is a highly regulated process. Animal pathogens use a length-control protein to produce T3SS needles of fixed length and then a second regulator, such as YopN in Yersinia spp, to mediate host contact-dependent effector delivery. For Pseudomonas syringae and other plant pathogens, regulation of the assembly process differs because the T3SS pilus must grow through variably thick plant cell walls before contacting the host plasma membrane. In this issue of Molecular Microbiology, Crabill et al. (2012) report evidence that the YopN homologue HrpJ is a multifunctional regulator of T3SS assembly in DC3000. A hrpJ mutant hyper-secretes pilus protein and no longer secretes four translocator proteins in culture, and it fails to inject effectors in planta. As with other proteins in this class, HrpJ is itself a T3SS substrate, but secretion-incompetent forms retain regulatory function. However, HrpJ is unusual in suppressing innate immune responses within host cells, as demonstrated with transgenic plants. The multiple capabilities of HrpJ appear to couple host contact sensing with pilus length control and translocator secretion while also contributing to immunity suppression early in the interaction.

Diverse interactions of five core type III effectors from Ralstonia solanacearum with plants.

Ralstonia solanacearum is a widespread plant bacterial pathogen that can launch a range of type III effectors (T3Es) to cause disease. In this study, we isolate a pathogenic R. solanacearum strain named P380 from tomato rhizosphere. Five out of 12 core T3Es of strain P380 are introduced into Pseudomonas syringae DC3000D36E separately to determine their functions in interacting with plants. DC3000D36E that harbors each effector suppresses FliC-triggered Pti5 and ACRE31 expression, ROS burst, and callose deposition. RipAE, RipU, and RipW elicit cell death as well as upregulate the MAPK cascades in Nicotiana benthamiana. The derivatives RipC1ΔDXDX(T/V) and RipWΔDKXXQ but not RipAEK310R fail to suppress ROS burst. Moreover, RipAEK310R and RipWΔDKXXQ retain the cell death elicitation ability. RipAE and RipW are associated with salicylic acid and jasmonic acid pathways, respectively. RipAE and RipAQ significantly promote the propagation of DC3000D36E in plants. The five core T3Es localize in diverse subcellular organelles of nucleus, plasma membrane, endoplasmic reticulum, and Golgi network. The suppressor of G2 allele of Skp1 is required for RipAE but not RipU-triggered cell death in N. benthamiana. These results indicate that the core T3Es in R. solanacearum play diverse roles in plant-pathogen interactions.

Duplicated Flagellins in Pseudomonas Divergently Contribute to Motility and Plant Immune Elicitation.

Flagellins are the main constituents of the flagellar filaments that provide bacterial motility, chemotactic ability, and host immune elicitation ability. Although the functions of flagellins have been extensively studied in bacteria with a single flagellin-encoding gene, the function of multiple flagellin-encoding genes in a single bacterial species is largely unknown. Here, the model plant-growth-promoting bacterium Pseudomonas kilonensis F113 was used to decipher the divergent functions of duplicated flagellins. We demonstrate that the two flagellins (FliC-1 and FliC-2) in 12 Pseudomonas strains, including F113, are evolutionarily distinct. Only the fliC-1 gene but not the fliC-2 gene in strain F113 is responsible for flagellar biogenesis, motility, and plant immune elicitation. The transcriptional expression of fliC-2 was significantly lower than that of fliC-1 in medium and in planta, most likely due to variations in promoter activity. In silico prediction revealed that all fliC-2 genes in the 12 Pseudomonas strains have a poorly conserved promoter motif. Compared to the Flg22-2 epitope (relative to FliC-2), Flg22-1 (relative to FliC-1) induced stronger FLAGELLIN SENSING 2 (FLS2)-mediated microbe-associated molecular pattern-triggered immunity and significantly inhibited plant root growth. A change in the 19th amino acid in Flg22-2 reduced its binding affinity to the FLS2/brassinosteroid insensitive 1-associated kinase 1 complex. Also, Flg22-2 epitopes in the other 11 Pseudomonas strains were presumed to have low binding affinity due to the same change in the 19th amino acid. These findings suggest that Pseudomonas has evolved duplicate flagellins, with only FliC-1 contributing to motility and plant immune elicitation. IMPORTANCE Flagellins have emerged as important microbial patterns. This work focuses on flagellin duplication in some plant-associated Pseudomonas. Our findings on the divergence of duplicated flagellins provide a conceptual framework for better understanding the functional determinant flagellin and its peptide in multiple-flagellin plant-growth-promoting rhizobacteria.

Pseudomonas syringae Type III Secretion Protein HrpP Manipulates Plant Immunity To Promote Infection.

The bacterial plant pathogen Pseudomonas syringae deploys a type III secretion system (T3SS) to deliver effector proteins into plant cells to facilitate infection, for which many effectors have been characterized for their interactions. However, few T3SS Hrp (hypersensitive response and pathogenicity) proteins from the T3SS secretion apparatus have been studied for their direct interactions with plants. Here, we show that the P. syringae pv. tomato DC3000 T3SS protein HrpP induces host cell death, suppresses pattern-triggered immunity (PTI), and restores the effector translocation ability of the hrpP mutant. The hrpP-transgenic Arabidopsis lines exhibited decreased PTI responses to flg22 and elf18 and enhanced disease susceptibility to P. syringae pv. tomato DC3000. Transcriptome analysis reveals that HrpP sensing activates salicylic acid (SA) signaling while suppressing jasmonic acid (JA) signaling, which correlates with increased SA accumulation and decreased JA biosynthesis. Both yeast two-hybrid and bimolecular fluorescence complementation assays show that HrpP interacts with mitogen-activated protein kinase kinase 2 (MKK2) on the plant membrane and in the nucleus. The HrpP truncation HrpP1-119, rather than HrpP1-101, retains the ability to interact with MKK2 and suppress PTI in plants. In contrast, HrpP1-101 continues to cause cell death and electrolyte leakage. MKK2 silencing compromises SA signaling but has no effect on cell death caused by HrpP. Overall, our work highlights that the P. syringae T3SS protein HrpP facilitates effector translocation and manipulates plant immunity to facilitate bacterial infection. IMPORTANCE The T3SS is required for the virulence of many Gram-negative bacterial pathogens of plants and animals. This study focuses on the sensing and function of the T3SS protein HrpP during plant interactions. Our findings show that HrpP and its N-terminal truncation HrpP1-119 can interact with MKK2, promote effector translocation, and manipulate plant immunity to facilitate bacterial infection, highlighting the P. syringae T3SS component involved in the fine-tuning of plant immunity.

Deciphering the rhizosphere bacteriome associated with biological control of tobacco black shank disease.

Introduction: The black shank disease seriously affects the health of tobacco plants. Conventional control methods have limitations in terms of effectiveness or economic aspects and cause public health concerns. Thus, biological control methods have come into the field, and microorganisms play a key role in suppressing tobacco black shank disease. Methods: In this study, we examined the impact of soil microbial community on black shank disease basing on the structural difference of bacterial communities in rhizosphere soils. We used Illumina sequencing to compare the bacterial community diversity and structure in different rhizosphere soil samples in terms of healthy tobacco, tobacco showing typical black shank symptoms, and tobacco treated with the biocontrol agent, Bacillus velezensis S719. Results: We found that Alphaproteobacteria in the biocontrol group, accounted for 27.2% of the ASVs, was the most abundant bacterial class among three groups. Heatmap and LEfSe analyses were done to determine the distinct bacterial genera in the three sample groups. For the healthy group, Pseudomonas was the most significant genus; for the diseased group, Stenotrophomonas exhibited the strongest enrichment trend, and Sphingomonas showed the highest linear discriminant analysis score, and was even more abundant than Bacillus; for the biocontrol group, Bacillus, and Gemmatimonas were the largely distributed genus. In addition, co-occurrence network analysis confirmed the abundance of taxa, and detected a recovery trend in the network topological parameters of the biocontrol group. Further functional prediction also provided a possible explanation for the bacterial community changes with related KEGG annotation terms. Discussion: These findings will improve our knowledge of plant-microbe interactions and the application of biocontrol agents to improve plant fitness, and may contribute to the selection of biocontrol strains.

Lysobacter changpingensis sp. nov., a novel species of the genus Lysobacter isolated from a rhizosphere soil of strawberry in China.

In the present work, we characterized in detail strain CM-3-T8T, which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8T were Gram-negative, non-spore-forming, aerobic, short rod. Growth occurred at 25-37 °C, pH 5.0-10.0, and in the presence of 0-8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CM-3-T8T formed a stable clade with Lysobacter soli DCY21T and Lysobacter panacisoli CJ29T, with the 16S rRNA gene sequence similarities of 98.91% and 98.50%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG-8 T and the two reference type strains listed above were 76.3%, 79.6%, and 34.3%, 27%, respectively. The DNA G + C content was 68.4% (mol/mol). The major cellular fatty acids were comprised of C15:0 iso (36.15%), C17:0 iso (8.40%), and C11:0 iso 3OH (8.28%). The major quinone system was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylethanolamine (PME), diphosphatidylglycerol (DPG), and aminophospholipid (APL). On the basis of phenotypic, genotypic, and phylogenetic evidence, strain CM-3-T8T (= ACCC 61714 T = JCM 34576 T) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed.

Dynamics of rice microbiomes reveal core vertically transmitted seed endophytes.

BACKGROUND: Plants and their associated microbiota constitute an assemblage of species known as holobionts. The plant seed microbiome plays an important role in nutrient uptake and stress attenuation. However, the core vertically transmitted endophytes remain largely unexplored. RESULTS: To gain valuable insights into the vertical transmission of rice seed core endophytes, we conducted a large-scale analysis of the microbiomes of two generations of six different rice varieties from five microhabitats (bulk soil, rhizosphere, root, stem, and seed) from four geographic locations. We showed that the microhabitat rather than the geographic location and rice variety was the primary driver of the rice microbiome assemblage. The diversity and network complexity of the rice-associated microbiome decreased steadily from far to near the roots, rice exterior to interior, and from belowground to aboveground niches. Remarkably, the microbiomes of the roots, stems, and seeds of the rice interior compartments were not greatly influenced by the external environment. The core bacterial endophytes of rice were primarily comprised of 14 amplicon sequence variants (ASVs), 10 of which, especially ASV_2 (Pantoea) and ASV_48 (Xanthomonas), were identified as potentially vertically transmitted taxa because they existed across generations, were rarely present in exterior rice microhabitats, and were frequently isolated from rice seeds. The genome sequences of Pantoea and Xanthomonas isolated from the parental and offspring seeds showed a high degree of average nucleotide and core protein identity, indicating vertical transmission of seed endophytes across generations. In silico prediction indicated that the seed endophytes Pantoea and Xanthomonas possessed streamlined genomes with short lengths, low-complexity metabolism, and various plant growth-promoting traits. We also found that all strains of Pantoea and Xanthomonas exhibited cellulase activity and produced indole-3-acetic acid. However, most strains exhibited insignificant antagonism to the major pathogens of rice, such as Magnaporthe oryzae and X. oryzae pv. oryzae. CONCLUSION: Overall, our study revealed that microhabitats, rather than site-specific environmental factors or host varieties, shape the rice microbiome. We discovered the vertically transmitted profiles and keystone taxa of the rice microbiome, which led to the isolation of culturable seed endophytes and investigation of their potential roles in plant-microbiome interactions. Our results provide insights on vertically transmitted microbiota and suggest new avenues for improving plant fitness via the manipulation of seed-associated microbiomes.  Video Abstract.

Bacterial effector HopF2 suppresses arabidopsis innate immunity at the plasma membrane.

Many bacterial pathogens inject a cocktail of effector proteins into host cells through type III secretion systems. These effectors act in concert to modulate host physiology and immune signaling, thereby promoting pathogenicity. In a search for additional Pseudomonas syringae effectors in suppressing plant innate immunity triggered by pathogen or microbe-associated molecular patterns (PAMPs or MAMPs), we identified P. syringae tomato DC3000 effector HopF2 as a potent suppressor of early immune-response gene transcription and mitogen-activated protein kinase (MAPK) signaling activated by multiple MAMPs, including bacterial flagellin, elongation factor Tu, peptidoglycan, lipopolysaccharide and HrpZ1 harpin, and fungal chitin. The conserved surface-exposed residues of HopF2 are essential for its MAMP suppression activity. HopF2 is targeted to the plant plasma membrane through a putative myristoylation site, and the membrane association appears to be required for its MAMP-suppression function. Expression of HopF2 in plants potently diminished the flagellin-induced phosphorylation of BIK1, a plasma membrane-associated cytoplasmic kinase that is rapidly phosphorylated within one minute upon flagellin perception. Thus, HopF2 likely intercepts MAMP signaling at the plasma membrane immediately of signal perception. Consistent with the potent suppression function of multiple MAMP signaling, expression of HopF2 in transgenic plants compromised plant nonhost immunity to bacteria P. syringae pv. Phaseolicola and plant immunity to the necrotrophic fungal pathogen Botrytis cinerea.

Characterization of the SPI-1 Type III Secretion System in Pseudomonas fluorescens 2P24.

Pseudomonas fluorescens 2P24 is a plant growth-promoting rhizobacterium (PGPR) isolated from wheat take-all decline soil. Genomic analysis of strain 2P24 revealed the presence of a complete SPI-1 type III secretion system (T3SS) gene cluster on the chromosome with an organization and orientation similar to the SPI-1 T3SS gene clusters of Salmonella enterica and P. kilonensis F113. Phylogenetic analysis revealed that the SPI-1 T3SS gene cluster of strain 2P24 might be obtained from Salmonella and Shigella by horizontal gene transfer. Two transcriptional regulator homologs of HilA and InvF were found from the SPI-1 T3SS gene cluster of strain 2P24. HilA regulated the expression of the structural genes positively, such as invG, sipB, sipD, prgI, and prgK. Prediction of transcriptional binding sites and RNA-seq analysis revealed 14 genes were up-regulated by InvF in strain 2P24. Exploring potential roles of SPI-1 T3SS revealed that it was not associated with motility. However, 2P24ΔinvF reduced resistance against Fusarium graminearum significantly. 2P24ΔhilA enhanced formation of biofilm significantly at 48 h. All three mutants 2P24ΔhilA, 2P24ΔinvF, and 2P24ΔinvE-C reduced the chemotactic responses to glucose significantly. Finally, the determination of SPI-1 mutants to trigger innate immunity in Nicotiana benthamiana showed that 2P24ΔinvE-C reduced the ability to induce the production of reactive oxygen species compared with the wild type strain 2P24.

Characterization of a Versatile Plant Growth-Promoting Rhizobacterium Pseudomonas mediterranea Strain S58.

Plant growth-promoting rhizobacterial strain S58 was isolated from the tobacco rhizosphere. It showed strong antagonism against a battery of plant pathogenic fungi and bacteria, and controlled wheat sharp eyespot and tobacco wildfire diseases efficiently. Further tests showed that strain S58 solubilized organic phosphate and produced siderophore, protease, ammonia, and indole-3-acetic acid. In Arabidopsis thaliana, it promoted plant growth and changed root system architecture by restricting the growth of primary roots and increasing lateral root numbers. We relied on morphological, biochemical, physiological characteristics, and molecular phylogenic analysis to identify strain S58 as Pseudomonas mediterranea. The complete genome of strain S58 has a single circular chromosome of 6,150,838 bp with a 61.06% G+C content. The bacterial genome contained 5,312 predicted genes with an average length of 992.90 bp. A genome analysis suggested that P. mediterranea S58 was a rich cyclic lipopeptide (CLP)-producing strain that possessed seven non-ribosomal peptide gene clusters for CLP synthesis. Leaf inoculation of the bacterial culture and supernatants triggered cell death-like immunity in tobacco. Quantitative real-time PCR assays showed that the strain S58 induced the expression of pattern-triggered immunity and cell death marker genes, but not jasmonic acid marker genes. The results suggested that P. mediterranea S58 is a novel, versatile plant growth-promoting agent with multiple beneficial traits for plants.

Genomic insights into a plant growth-promoting Pseudomonas koreensis strain with cyclic lipopeptide-mediated antifungal activity.

Strain S150 was isolated from the tobacco rhizosphere as a plant growth-promoting rhizobacterium. It increased plant fresh weight significantly and lateral root development, and it antagonized plant pathogenic fungi but not phytobacteria. Further tests showed that strain S150 solubilized organic phosphate and produced ammonia, siderophore, protease, amylase, and cellulase, but it did not produce indole-3-acetic acid. Using morphology, physiological characteristics, and multi-locus sequence analysis, strain S150 was identified as Pseudomonas koreensis. The complete genome of strain S150 was sequenced, and it showed a single circular chromosome of 6,304,843 bp with a 61.09% G + C content. The bacterial genome contained 5,454 predicted genes that occupied 87.7% of the genome. Venn diagrams of the identified orthologous clusters of P. koreensis S150 with the other three sequenced P. koreensis strains revealed up to 4,167 homologous gene clusters that were shared among them, and 21 orthologous clusters were only present in the genome of strain S150. Genome mining of the bacterium P. koreensis S150 showed that the strain possessed 10 biosynthetic gene clusters for secondary metabolites, which included four clusters of non-ribosomal peptide synthetases (NRPSs) involved in the biosynthesis of cyclic lipopeptides (CLPs). One of the NRPSs possibly encoded lokisin, a cyclic lipopeptide produced by fluorescent Pseudomonas. Genomic mutation of the lokA gene, which is one of the three structural NRPS genes for lokisin in strain S150, led to a deficiency in fungal antagonism that could be restored fully by gene complementation. The results suggested that P. koreensis S150 is a novel plant growth-promoting agent with specific cyclic lipopeptides and contains a lokisin-encoding gene cluster that is dominant against plant fungal pathogens.