Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

Simultaneous removal of aliphatic and aromatic crude oil hydrocarbons by Pantoea agglomerans isolated from petroleum-contaminated soil in the west of Iran.

Hydrocarbons are considered as one of the most common and harmful environmental pollutants affecting human health and the environment. Bioremediation as an environmentally friendly, highly efficient, and cost-effective method in remediating oil-contaminated environments has been interesting in recent decades. In this study, hydrocarbon degrader bacterial strains were isolated from the highly petroleum-contaminated soils in the Dehloran oil field in the west of Iran. Out of 37 isolates, 15 can grow on M9 agar medium that contains 1.5 g L-1 of crude oil as the sole carbon source. The morphological, biochemical, and 16SrRNA sequencing analyses were performed for the isolates. The choosing of the isolates as the hydrocarbon degrader was examined by evaluating the efficacy of their crude oil removal at a concentration of 10 g L-1 in an aqueous medium. The results showed that five isolates belonging to Pseudomonas sp., Pseudomonas oryzihabitans, Roseomonas aestuarii, Pantoea agglomerans, and Arthrobacter sp. had a hyper hydrocarbon-degrading activity and they could remove more than 85% of the total petroleum hydrocarbon (TPH) after 96 h. The highest TPH removal of about 95.75% and biodegradation rate of 0.0997 g L-1 h-1 was observed for P. agglomerans. The gas chromatography-mass spectroscopy (GC-MS) analysis was performed during the biodegradation process by P. agglomerans to detect the degradation intermediates and final products. The results confirmed the presence of intermediates such as alcohols and fatty acids in the terminal oxidation pathway of alkanes in this biodegradation process. A promising P. agglomerans NB391 strain can remove aliphatic and aromatic hydrocarbons simultaneously.

Pantoea trifolii sp. nov., a novel bacterium isolated from Trifolium rubens root nodules.

A novel bacterium, designated strain MMK2T, was isolated from a surface-sterilised root nodule of a Trifolium rubens plant growing in south-eastern Poland. Cells were Gram negative, non-spore forming and rod shaped. The strain had the highest 16S rRNA gene sequence similarity with P. endophytica (99.4%), P. leporis (99.4%) P. rwandensis (98.8%) and P. rodasii (98.45%). Phylogenomic analysis clearly showed that strain MMK2T and an additional strain, MMK3, should reside in the genus Pantoea and that they were most closely related to P. endophytica and P. leporis. Genome comparisons showed that the novel strain shared 82.96-93.50% average nucleotide identity and 26.2-53. 2% digital DNA:DNA hybridization with closely related species. Both strains produced siderophores and were able to solubilise phosphates. The MMK2T strain was also able to produce indole-3-acetic acid. The tested strains differed in their antimicrobial activity, but both were able to inhibit the growth of Sclerotinia sclerotiorum 10Ss01. Based on the results of the phenotypic, phylogenomic, genomic and chemotaxonomic analyses, strains MMK2T and MMK3 belong to a novel species in the genus Pantoea for which the name Pantoea trifolii sp. nov. is proposed with the type strain MMK2T (= DSM 115063T = LMG 33049T).

Rice foliar-adapted Pantoea species: Promising microbial biostimulants enhancing rice resilience against foliar pathogens, Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae.

Foliar fungal blast and bacterial leaf blight have significant impacts on rice production, and their management through host resistance and agrochemicals has proven inadequate. To achieve their sustainable management, innovative approaches like leveraging the foliar microbiome, which collaborates with plants and competes against pathogens, are essential. In our study, we isolated three Pantoea strains (P. agglomerans Os-Ep-PPA-1b, P. vagans Os-Ep-PPA-3b, and P. deleyi Os-Ep-VPA-9a) from the rice phylloplane. These isolates exhibited antimicrobial action through their metabolome and volatilome, while also promoting rice growth. Our analysis, using Gas Chromatography-Mass Spectrometry (GC-MS), revealed the presence of various antimicrobial compounds such as esters and fatty acids produced by these Pantoea isolates. Inoculating rice seedlings with P. agglomerans and P. vagans led to increased root and shoot growth. Additionally, bacterized seedlings displayed enhanced immunocompetence, as evidenced by upregulated expressions of defense genes (OsEDS1, OsFLS2, OsPDF2.2, OsACO4, OsICS OsPR1a, OsNPR1.3, OsPAD4, OsCERK1.1), along with heightened activities of defense enzymes like Polyphenol Oxidase and Peroxidase. These plants also exhibited elevated levels of total phenols. In field trials, the Pantoea isolates contributed to improved plant growth, exemplified by increased flag-leaf length, panicle number, and grains per panicle, while simultaneously reducing the incidence of chaffy grains. Hypersensitivity assays performed on a model plant, tobacco, confirmed the non-pathogenic nature of these Pantoea isolates. In summary, our study underscores the potential of Pantoea bacteria in combatting rice foliar diseases. Coupled with their remarkable growth-promoting and biostimulant capabilities, these findings position Pantoea as promising agents for enhancing rice cultivation.

Pantailocins: phage-derived bacteriocins from Pantoea ananatis and Pantoea stewartii subsp. indologenes.

IMPORTANCE: Phage-derived bacteriocins (tailocins) are ribosomally synthesized structures produced by bacteria in order to provide advantages against competing strains under natural conditions. Tailocins are highly specific in their target range and have proven to be effective for the prevention and/or treatment of bacterial diseases under clinical and agricultural settings. We describe the discovery and characterization of a new tailocin locus encoded within genomes of Pantoea ananatis and Pantoea stewartii subsp. indologenes, which may enable the development of tailocins as preventative treatments against phytopathogenic infection by these species.

A discovery down under: decoding the draft genome sequence of Pantoea stewartii from Australia's Critically Endangered western ground parrot/kyloring (Pezoporus flaviventris).

Pantoea stewartii, a plant pathogen, is primarily transmitted through contaminated seeds and insect vectors, with the corn flea beetle (Chaetocnema pulicaria) being the primary carrier. P. stewartii is a bacterium belonging to the order Enterobacterales and can lead to crop diseases that have a significant economic impact worldwide. Due to its high potential for spread, P. stewartii is classified as a quarantine organism in numerous countries. Despite its impact on agriculture, the limited genome sequences of P. stewartii hamper understanding of its pathogenicity and host specificity, and the development of effective control strategies. In this study, a P. stewartii strain (C10109_Jinnung) was discovered in the faecal matter of the Critically Endangered western ground parrot/kyloring (Pezoporus flaviventris) in Australia, which to our knowledge is the first reported P. stewartii genome from a bird source. Whole-genome sequencing and phylogenomic analysis of strain C10109_Jinnung, obtained from a captive psittacine, provides new insights into the genetic diversity and potential transmission route for the spread of P. stewartii beyond insects and plants, where P. stewartii is typically studied. Our findings provide new insights into the potential transmission route for spread of P. stewartii and expand the known transmission agents beyond insects and plants. Expanding the catalogue of P. stewartii genomes is fundamental to improving understanding of the pathogenicity, evolution and dissemination, and to develop effective control strategies to reduce the substantial economic losses associated with P. stewartii in various crops and the potential impact of endangered animal species.

A replica plating method for efficient, high-throughput screening of antibiotic gene clusters in bacteria uncovers a holomycin-like cluster in the clinical isolate, Pantoea agglomerans 20KB447973.

Bacterial natural products remain a major untapped source for novel antimicrobial scaffolds. Many of these products are encoded by biosynthetic gene clusters (BGCs), which can be identified using functional genomics. We developed a replica-plating approach to quickly screen for antibiotic production mutants from transposon mutant libraries and identify candidate antibiotic BGCs. In this technique, filter paper is used to transfer up to 200 mutants simultaneously onto a soft agar overlay or spread plate containing a target microbe to identify antibiotic-production mutants. These mutants can then be analyzed to identify disrupted genes and antibiotic BGCs. We first tested and optimized this technique by screening for previously characterized BGCs in Pantoea. We then applied the technique to uncover the gene cluster responsible for the production of an unknown broad-spectrum antibiotic from P. agglomerans 20KB447973, which we call Pantoea Natural Product 5 (PNP-5). Analysis of the predicted gene cluster for PNP-5 showed similarity to previously identified gene clusters for the broad-spectrum dithiolopyrrolone antibiotic, holomycin. Analysis of the spectrum of activity of PNP-5 showed activity against members of the Enterobacteriaceae, Erwiniaceae, and Streptococcaceae, including clinically relevant pathogens such as Klebsiella sp. and Escherichia coli. We also identified the production of a second antibiotic, pantocin A. Our findings demonstrate the utility of our replica-plating mutant transfer method in exploring unknown antibiotic BGCs. Adoption of this technique may accelerate the identification of potentially novel antimicrobial BGCs within strain collections, advancing the search for novel antimicrobials that can be used to treat multi-drug resistant infections.

Analysis of key genes for the survival of Pantoea agglomerans under nutritional stress.

The absolute amount of nutrients on plant leaves is usually low, and the growth of epiphytic bacteria is typically limited by nutrient content. Thus, is of great significance to study the survival mechanism of epiphytes under nutritional stress for plant disease control. In this paper, Pantoea agglomerans CHTF15 isolated from walnut leaves was used to detect the key genes for the survival of the bacterium under simulated nutrient stress in artificial medium. Genome sequencing was combined with transposon insertion sequencing (Tn-seq) for the detection technique. A total of 105 essential genes were screened from the whole genome. The genes were mainly related to the nucleotide metabolism, protein metabolism, biological oxidation and the gene repair of bacteria analyzed by gene ontology (GO) enrichment analysis. Volcano map analysis demonstrated that the functions of the 15 genes with the most significant differences were generally related to the synthesis of amino acids or proteins, the nucleotide metabolism and homologous recombination and repair. Competitive index analysis revealed that the deletion of the genes dksA and epmA regulating protein synthesis, the gene ribB involved in the nucleotide metabolism and the gene xerD involved in recombination repair induced a significant reduction in the survival ability of the corresponding mutants in the 0.10 % YEP medium and the walnut leaf surface. The results act as a foundation for further in-depth research on the infection process and the mechanisms of pathogenic bacteria.

Siderophore-producing Pantoea ferrattrahens sp. nov. isolated from a clinical specimen and Pantoea ferramans sp. nov. isolated from soil at the bottom of a pond.

Two Gram-negative facultative anaerobes were isolated from a sepsis patient with pancreatic cancer (strain PAGU 2156T ) and soil at the bottom of a pond (strain PAGU 2198T ), respectively. These two strains formed haloes around the colonies on chrome azurol S agar plates, indicating the production of siderophores. Two isolates assigned to the genus Pantoea based on the 16S rRNA gene were differentiated from established species by using polymorphic taxonomies. Phylogenetic analysis using four housekeeping genes (gyrB, rpoB, atpD, and infB) showed that strain PAGU 2156T is closely related to Pantoea cypripedii LMG 2657T (89.9%) or Pantoea septica LMG 5345T (95.7%). Meanwhile, strain PAGU 2198T formed a single clade with Pantoea rodasii DSM 26611T (93.6%) and Pantoea rwandensis DSM 105076T (93.3%). The average nucleotide identity values obtained from the draft genome assembly showed ≤90.2% between strain PAGU 2156T and closely related species and ≤81.5% between strain PAGU 2198T and closely related species. Based on various phenotypes, biochemical properties, and whole-cell fatty acid composition compared with related species, it was concluded that each strain should be classified as a new species of the genus Pantoea. In this manuscript, Pantoea ferrattrahens sp. nov. and Pantoea ferramans sp. nov. with strain PAGU 2156T (=NBRC 115930T = CCUG 76757T ) and strain PAGU 2198T (=NBRC 114265T = CCUG 75151T ) are proposed as each type strain.

Transcriptional Profiling and Transposon Mutagenesis Study of the Endophyte Pantoea eucalypti FBS135 Adapting to Nitrogen Starvation.

The research on plant endophytes has been drawing a lot of attention in recent years. Pantoea belongs to a group of endophytes with plant growth-promoting activity and has been widely used in agricultural fields. In our earlier studies, Pantoea eucalypti FBS135 was isolated from healthy-growing Pinus massoniana and was able to promote pine growth. P. eucalypti FBS135 can grow under extremely low nitrogen conditions. To understand the mechanism of the low-nitrogen tolerance of this bacterium, the transcriptome of FBS135 in the absence of nitrogen was examined in this study. We found that FBS135 actively regulates its gene expression in response to nitrogen deficiency. Nearly half of the number (4475) of genes in FBS135 were differentially expressed under this condition, mostly downregulated, while it significantly upregulated many transportation-associated genes and some nitrogen metabolism-related genes. In the downregulated genes, the ribosome pathway-related ones were significantly enriched. Meanwhile, we constructed a Tn5 transposon library of FBS135, from which four genes involved in low-nitrogen tolerance were screened out, including the gene for the host-specific protein J, RNA polymerase σ factor RpoS, phosphoribosamine-glycine ligase, and serine acetyltransferase. Functional analysis of the genes revealed their potential roles in the adaptation to nitrogen limitation. The results obtained in this work shed light on the mechanism of endophytes represented by P. eucalypti FBS135, at the overall transcriptional level, to an environmentally limited nitrogen supply and provided a basis for further investigation on this topic.

Pantoea leporis sp. nov., isolated from the faecal material of a rabbit.

A facultative anaerobic, Gram-stain-negative rod-shaped bacterium, designated RT, was isolated from the faecal material of a rabbit (Sylvilagus floridanus). The strain could not be identified using an MALDI Biotyper sirius CA System. The closest matches based on the Bruker library were members of the genera Citrobacter and Pantoea. However, the score value was in the range of no organism identification possible. Based on pairwise of 16S rRNA gene sequence analysis, the isolate was found to be a member of the family Erwiniaceae. The highest sequence similarities were found to the sequences of Pantoea rodasii LMG 26273T (98.7 %), Leclercia adecarboxylata NBRC 102595T (98.5 %) and Enterobacter huaxiensis 090008T (98.4 %). Phylogenetic and whole genome analysis demonstrated that strain RT represents a novel species within the genus Pantoea. The predominant cellular fatty acids of strain RT were C16 : 0 and products present in summed feature 2 (C12 : 0) aldehyde, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). In silico genome analysis showed the presence of enzymes required for production of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine. The G+C content determined from the genome was 54.94 mol %. Based on biochemical, phylogenetic, genotypic and chemotaxonomic criteria, the isolate represents a novel species of the genus Pantoea for which the name Pantoea leporis sp. nov. is proposed. The type strain is strain RT (=CCUG 76269T=ATCC TSD-291T).

High Prevalence of Pantoea spp. in Microbiota Associated with the Sorghum Plant Bug Stenotus rubrovittatus (Heteroptera: Miridae).

The sorghum plant bug, Stenotus rubrovittatus (order Heteroptera: family Miridae), is a notorious insect pest in Japan that causes pecky rice. In the present study, we sampled this insect pest in the northern part of Honshu Island in Japan and investigated its associated microbiota. The results obtained showed that Pantoea dominated the associated microbiota and was the sole genus detected in all samples. The dominant Pantoea were phylogenetically close to rice pathogens. The present results suggest that the sorghum plant bug needs to be regarded and controlled not only as a notorious insect pest, but also as a potential vector of rice pathogenic Pantoea spp.

Genomic characterization of Pantoea anthophila strain UI705 causing urinary tract infections in China.

Introduction: Pantoea anthophila (P. anthophila) is a Gram-negative bacterium initially isolated from Impatiens balsamina in India. P. anthophila has been characterized with low pathogenicity, and no human infections caused by this organism have been reported yet. We report the first case of urinary tract infection caused by P. anthophila in a 73-year-old man after bladder cancer surgery. Methods: The bacterial isolate gained from urine was named UI705 and identified as P. anthophila by MALDI-TOF mass spectrometry. The genome sequencing and analysis were performed to further characterize the pathogenesis of the clinical isolate. Result and discussion: To the best of our knowledge, this is the first report of human infection caused by P. anthophila in China. The draft genome sequence of P. anthophila UI705 provides a fundamental resource for subsequent investigation of its virulence factors, antibiotic resistance, host-pathogen interactions, and comparative genomics of genus Pantoea.

A new l-serine binding orphan SerBP affects indole synthesis in Pantoea ananatis.

Indole is traditionally known as a metabolite of l-tryptophan and now as an important signaling molecule in bacteria, however, the understanding of its upstream synthesis regulation is very limited. Pantoea ananatis YJ76, a predominant diazotrophic endophyte isolated from rice (Oryza sativa), can produce indole to regulate various physiological and biochemical behaviors. We constructed a mutant library of YJ76 using the mTn5 transposon insertion mutation method, from which an indole-deficient mutant was screened out. Via high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR), the transposon was determined to be inserted in a gene (RefSeq: WP014605468.1) of unknown function that is highly conserved at the intraspecific level. Bioinformatics analysis implied that the protein (Protein ID: WP089517194.1) encoded by the mutant gene is most likely to be a new orphan substrate-binding protein (SBP) for amino acid ABC transporters. Amino acid supplement cultivation experiments and surface plasmon resonance revealed that the protein could bind to l-serine (KD = 6.149 × 10-5 M). Therefore, the SBP was named as SerBP. This is the first case that a SBP responds to l-serine ABC transports. As a precursor of indole synthesis, the transmembrane transported l-serine was directly correlated with indole signal production and the mutation of serBP gene weakened the resistance of YJ76 to antibiotics, alkali, heavy metals, and starvation. This study provided a new paradigm for exploring the upstream regulatory pathway for indole synthesis of bacteria.

Contribution of Native Plasmids of Pantoea vagans C9-1 to Epiphytic Fitness and Fire Blight Management on Apple and Pear Flowers and Fruits.

Pantoea vagans C9-1 (C9-1) is a biological control bacterium that is applied to apple and pear trees during bloom for suppression of fire blight, caused by Erwinia amylovora. Strain C9-1 has three megaplasmids: pPag1, pPag2, and pPag3. Prior bioinformatic studies predicted these megaplasmids have a role in environmental fitness and/or biocontrol efficacy. Plasmid pPag3 is part of the large Pantoea plasmid (LPP-1) group that is present in all Pantoea spp. and has been hypothesized to contribute to environmental colonization and persistence, while pPag2 is less common. We assessed fitness of C9-1 derivatives cured of pPag2 and/or pPag3 on pear and apple flowers and fruit in experimental orchards. We also assessed the ability of a C9-1 derivative lacking pPag3 to reduce populations of E. amylovora on flowers and disease incidence. Previously, we determined that tolerance to stresses imposed in vitro was compromised in derivatives of C9-1 lacking pPag2 and/or pPag3; however, in this study, the loss of pPag2 and/or pPag3 did not consistently reduce the fitness of C9-1 on flowers in orchards. Over the summer, pPag3 contributed to survival of C9-1 on developing apple and pear fruit in two of five trials, whereas loss of pPag2 did not significantly affect survival of C9-1. We also found that loss of pPag3 did not affect C9-1's ability to reduce E. amylovora populations or fire blight incidence on apple flowers. Our findings partially support prior hypotheses that LPP-1 in Pantoea species contributes to persistence on plant surfaces but questions whether LPP-1 facilitates host colonization.

Biocontrol potential of actinobacteria against Pantoea ananatis, the causal agent of maize white spot disease.

Pantoea ananatis is the causal agent of maize white spot, a foliar disease responsible for significant maize yield reduction worldwide, especially in Brazil. In general, the maize foliar diseases control involves the adoption of resistant genotypes and pesticides application. However, the use of agrochemicals can significantly cause increase production costs, damage to human health and negative environmental impacts. In this sense, the use of biological control agents has been considered among the most promising eco-friendly technologies for sustainable agriculture. Actinobacteria, particularly of Streptomyces genus, has been widely recognized as agroindustrially important microorganism due to its potential in producing diverse range of secondary metabolites, including antibiotics and enzymes. Thus, the aim of this work is to characterize and to evaluate the potential of soil actinobacteria for P. ananatis control. We observed that 59 actinobacteria strains (85%) exhibited proteolytic or chitinolytic activity. Only the strains Streptomyces pseudovenezuelae ACSL 470, that also exhibited high proteolytic activity, S. novaecaesareae ACSL 432 and S. laculatispora ACP 35 demonstrated high or moderate antagonist activity in vitro against P. ananatis. Temporal analysis of metabolites produced by these strains growth in different liquid media indicated greater antibacterial activity at 72 h. In this condition, chromatographic and mass spectrometry analysis revealed that S. pseudovenezuelae ACSL 470 strain produced neomycin, an aminoglycoside antibiotic that displayed high bactericidal activity in vitro against P. ananatis. This is the first report of actinobacteria acting as potential microbial antagonists for P. ananatis control. Further studies are needed to determine the control efficacy of maize white spot disease by Streptomyces strains or their metabolites in greenhouse and field conditions.

Diverse Virulence Attributes of Pantoea alfalfae sp. nov. CQ10 Responsible for Bacterial Leaf Blight in Alfalfa Revealed by Genomic Analysis.

Alfalfa is widely grown worldwide for its excellent nutritional value. Pantoea species living in alfalfa seeds can easily spread over great distances with frequent trade. However, the pathogenic properties of this dangerous hitchhiker on alfalfa have not been evaluated. Here, we identified the taxonomic status of Pantoea strain CQ10 isolated from the interior of alfalfa seeds based on the whole genome sequence. The diverse virulence attributes of strain CQ10 during host infection were characterized through pathogenicity assays and functional and genomic analyses. We report that strain CQ10 belongs to a novel species in the genus Pantoea, which was phylogenetically close to Pantoea vagans and Pantoea agglomerans. Strain CQ10 caused bacterial leaf blight of alfalfa after inoculation from the roots. We found that strain CQ10 possesses a large number of pathogenic genes involved in shaping the virulence properties during bacteria-host interactions, including motility, biofilm, type VI secretion system, and nutrient acquisition. Compared with P. vagans and P. agglomerans, the unique virulence factors of strain CQ10 were mainly involved in motility and biofilm, which were confirmed by in vitro experiments. Taken together, our results suggest that strain CQ10 is the first Pantoea species to infect alfalfa, and it possesses diverse virulence attributes among which motility and biofilm may be the best weapons.

Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

Pantoea Bacteria Isolated from Three Thrips (Frankliniella occidentalis, Frankliniella intonsa, and Thrips tabaci) in Korea and Their Symbiotic Roles in Host Insect Development.

Gut symbionts play crucial roles in host development by producing nutrients and defending against pathogens. Phloem-feeding insects in particular lack essential nutrients in their diets, and thus, gut symbionts are required for their development. Gram-negative Pantoea spp. are known to be symbiotic to the western flower thrips (Frankliniella occidentalis). However, their bacterial characteristics have not been thoroughly investigated. In this study, we isolated three different bacteria (BFoK1, BFiK1, and BTtK1) from F. occidentalis, F. intonsa, and T. tabaci. The bacterial isolates of all three species contained Pantoea spp. Their 16S rRNA sequences indicated that BFoK1 and BTtK1 were similar to P. agglomerans, while BFiK1 was similar to P. dispersa. These predictions were supported by the biochemical characteristics assessed by fatty acid composition and organic carbon utilization. In the bacterial morphological analysis, BFoK1 and BTtK1 were distinct from BFiK1. All these bacteria were relatively resistant to tetracycline compared to ampicillin and kanamycin, in which BFoK1 and BTtK1 were different from BFiK1. Feeding ampicillin (100,000 ppm) reduced the bacterial density in thrips and retarded the development of F. occidentalis. The addition of BFoK1 bacteria, however, rescued the retarded development. These findings indicate that Pantoea bacteria are symbionts to different species of thrips.