Strain R1 Research Articles

Study on a novel immobilized microbe pellets constructed with Alcaligenes sp. R3 and its ability to remove tetracycline

Microbial technology provides a feasible and effective means for removing tetracycline from water bodies. In this study, a novel tetracycline removal strain was isolated from pig manure and identified as an Alcaligenes sp. The effects of inoculation dose, initial tetracycline concentration, pH and temperature on the tetracycline removal efficiencies of the Alcaligenes sp. R3 strain were investigated. Response surface methodology (RSM) using a Box-Behnken design demonstrated that the optimal conditions for the temperature, pH, inoculation dose of Alcaligenes sp. R3 and initial tetracycline concentration were 32.55 °C, 8.83, 17.32% and 54.58 mg/L, respectively. Alcaligenes sp. R3 could remove 71.62% of tetracycline at most. To improve the ability of Alcaligenes sp. R3 to remove tetracycline, it was immobilized with carboxymethylcellulose (CMC) and polydopamine (PDA) to construct immobilized microbe pellets (IMPs); scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) of the IMPs showed that the CMC/PDA exhibited good adsorption ability and biocompatibility and could thus be used as a carrier of the living cells. The maximum tetracycline removal percentage of the IMPs was 91.16%, indicating that the ability of the IMPs to remove tetracycline was superior to that of free cells. Through immobilization with CMC/PDA, IMPs could remove tetracycline more effectively and had better environmental tolerance than free Alcaligenes sp. R3. The identification of possible biodegradation products indicated that the degradation pathway of strain R3 was novel and CMC/PDA only adsorbed tetracycline but not have degradation effect on it.

Evaluation of Haloferax mediterranei Strain R4 Capabilities for Cadmium Removal from Brines.

Haloferax mediterranei has revealed a high bioremediation potential for several inorganic anions (e.g., nitrates and nitrites) and metals from hypersaline waters and brines. However, it is unclear, to date, whether this microorganism allows Cd (II) bioremediation. Consequently, the main objective of this work was to assess the Cd (II) bioremediation potential of Hfx. mediterranei R4. To this end, Hfx. mediterranei cell growth rate and metal bioaccumulation were investigated using different culture media (complex, CM, and defined medium, DM) containing Cd (II) up to 1 mM. In addition, the elemental profile of the biomass (i.e., Al, Ba, Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Sr and Zn) has also been monitored to gain insight into the metabolic processes that may be taking place at the intracellular level for Cd (II) removal. Because of the formation of CdS precipitate, CM is not a suitable culture media for evaluating Cd bioremediation since metal concentration could not be appropriately controlled. When operating in DM, it was observed that the cell doubling time increases three times in the presence of Cd (II). Hfx. mediterranei can bioaccumulate Cd, showing the highest significant accumulation at concentrations of 0.4 mM (108 ± 12 mg Cd/g dry tissue). Finally, the presence of Cd (II) affects the content of K, Mg, Mn and Zn in the biomass, by increasing K levels up to 27 ± 18% and Mn up to 310 ± 140% and reducing Mg levels up to 55 ± 36% and Zn up to 30 ± 4%. These results suggest that different mechanisms are involved in Cd (II) tolerance by Hfx. mediterranei, resulting in increasing the cell concentration of stress-tolerant elements in the biomass (K and Mn), while lowering the concentration of elements which Cd (II) competes with (Mg and Zn), and that all affects the physiological response of the organism by decreasing its growth rate.

Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering.

In this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 strain was constructed by the overexpression of zwf harboring the constructed plasmid pAC-Z in the R1 strain. Thus, the level of riboflavin in the R2 strain increased to 319.01 ± 20.65 mg/l (74.66% increase). To further enhance ribB transcript levels and riboflavin production, the FMN riboswitch was deleted from E. coli BL21 with CRISPR/Cas9 to generate the R3 strain. The R4 strain was constructed by cotransforming pET-AE and pAC-Z into the R3 strain. Compared to those of E. coli BL21, the ribB transcript levels of R2 and R4 improved 2.78 and 3.05-fold, respectively. The R4 strain accumulated 437.58 ± 14.36 mg/l riboflavin, increasing by 37.17% compared to the R2 strain. These results suggest that the deletion of the FMN riboswitch can improve the transcript level of ribB and facilitate riboflavin production. A riboflavin titer of 611.22 ± 11.25 mg/l was achieved under the optimal fermentation conditions. Ultimately, 1574.60 ± 109.32 mg/l riboflavin was produced through fed-batch fermentation with 40 g/l glucose. This study contributes to the industrial production of riboflavin by the recombinant E. coli BL21.

The balance between gyrase and topoisomerase I activities determines levels of supercoiling, nucleoid compaction, and viability in bacteria.

Two enzymes are responsible for maintaining supercoiling in the human pathogen Streptococcus pneumoniae, gyrase (GyrA2GyrB2) and topoisomerase I. To attain diverse levels of topoisomerase I (TopoI, encoded by topA), two isogenic strains derived from wild-type strain R6 were constructed: PZn topA, carrying an ectopic topA copy under the control of the ZnSO4-regulated PZn promoter and its derivative ΔtopAPZn topA, which carries a topA deletion at its native chromosomal location. We estimated the number of TopoI and GyrA molecules per cell by using Western-blot and CFUs counting, and correlated these values with supercoiling levels. Supercoiling was estimated in two ways. We used classical 2D-agarose gel electrophoresis of plasmid topoisomers to determine supercoiling density (σ) and we measured compaction of nucleoids using for the first time super-resolution confocal microscopy. Notably, we observed a good correlation between both supercoiling calculations. In R6, with σ = -0.057, the average number of GyrA molecules per cell (2,184) was higher than that of TopoI (1,432), being the GyrA:TopoI proportion of 1:0.65. In ΔtopAPZn topA, the number of TopoI molecules depended, as expected, on ZnSO4 concentration in the culture media, being the proportions of GyrA:TopoI molecules in 75, 150, and 300 μM ZnSO4 of 1:0.43, 1:0.47, and 1:0.63, respectively, which allowed normal supercoiling and growth. However, in the absence of ZnSO4, a higher GyrA:TopoI ratio (1:0.09) caused hyper-supercoiling (σ = -0.086) and lethality. Likewise, growth of ΔtopAPZn topA in the absence of ZnSO4 was restored when gyrase was inhibited with novobiocin, coincidentally with the resolution of hyper-supercoiling (σ change from -0.080 to -0.068). Given that TopoI is a monomer and two molecules of GyrA are present in the gyrase heterotetramer, the gyrase:TopoI enzymes proportion would be 1:1.30 (wild type R6) or of 1:1.26-0.86 (ΔtopAPZn topA under viable conditions). Higher proportions, such as 1:0.18 observed in ΔtopAPZn topA in the absence of ZnSO4 yielded to hyper-supercoiling and lethality. These results support a role of the equilibrium between gyrase and TopoI activities in supercoiling maintenance, nucleoid compaction, and viability. Our results shed new light on the mechanism of action of topoisomerase-targeting antibiotics, paving the way for the use of combination therapies.

Identification of a Phylogenetically Divergent Vanillate O-Demethylase from Rhodococcus ruber R1 Supporting Growth on Meta-Methoxylated Aromatic Acids.

Rieske-type two-component vanillate O-demethylases (VanODs) catalyze conversion of the lignin-derived monomer vanillate into protocatechuate in several bacterial species. Currently, VanODs have received attention because of the demand of effective lignin valorization technologies, since these enzymes own the potential to catalyze methoxy group demethylation of distinct lignin monomers. In this work, we identified a phylogenetically divergent VanOD from Rhodococcus ruber R1, only distantly related to previously described homologues and whose presence, along with a 3-hydroxybenzoate/gentisate pathway, correlated with the ability to grow on other meta-methoxylated aromatics, such as 3-methoxybenzoate and 5-methoxysalicylate. The complementation of catabolic abilities by heterologous expression in a host strain unable to grow on vanillate, and subsequent resting cell assays, suggest that the vanAB genes of R1 strain encode a proficient VanOD acting on different vanillate-like substrates; and also revealed that a methoxy group in the meta position and a carboxylic acid moiety in the aromatic ring are key for substrate recognition. Phylogenetic analysis of the oxygenase subunit of bacterial VanODs revealed three divergent groups constituted by homologues found in Proteobacteria (Type I), Actinobacteria (Type II), or Proteobacteria/Actinobacteria (Type III) in which the R1 VanOD is placed. These results suggest that VanOD from R1 strain, and its type III homologues, expand the range of methoxylated aromatics used as substrates by bacteria.

Invasion and Colonization of Pathogenic Fusarium oxysporum R1 in Crocus sativus L. during Corm Rot Disease Progression.

The corm rot of saffron caused by Fusarium oxysporum (Fox) has been reported to be the most destructive fungal disease of the herb globally. The pathogen, Fusarium oxysporum R1 (Fox R1) isolated by our group from Kashmir, India, was found to be different from Fusarium oxysporum f.sp. gladioli commonly reported corm rot agent of saffron. In the present study, Fox R1 was further characterized using housekeeping genes and pathogenicity tests, as Fusarium oxysporum R1 f.sp. iridacearum race 4. Though Fox R1 invaded the saffron plant through both corm and roots, the corm was found to be the preferred site of infection. In addition, the route of pathogen movement wastracked by monitoring visual symptoms, semi-quantitative PCR, quantitative-PCR (q-PCR), real-time imaging of egfp-tagged Fusarium oxysporum R1, and Fox R1 load quantification. This study is the first study of its kind on the bidirectional pathogenesis from corm to roots and vice-versa, as the literature only reports unidirectional upward movement from roots to other parts of the plant. In addition, the colonization pattern of Fox R1 in saffron corms and roots was studied. The present study involved a systematic elucidation of the mode and mechanism of pathogenesis in the saffron Fusarium oxysporum strain R1 pathosystem.

Screening and identification of lactic acid bacteria with antimicrobial abilities for aquaculture pathogens in vitro

This study aimed to evaluate the antibacterial properties of nine lactic acid bacterial strains. The agar diffusion method (Oxford cup method) was used to assess the antimicrobial activity against pathogenic bacteria in aquaculture. The results showed that all selected strains inhibited the growth of Photobacterium damsel, Vibrio alginolyticus, Listonella anguillarum, Edwardsiella ictaluri, Aeromonas hydrophila, V. arahaemolyticus, Edwardsiella tarda and V. harveyi, but did not inhibit the growth of Metschnikowia bicuspidata. Among these strains of lactic acid bacteria, r1 was selected for its obvious antibacterial activity against eight kinds of pathogenic bacteria by 96-well plate method. Its inhibitory rate reached 96%, and it could inhibit the growth of six pathogenic bacteria at 121℃, 20min and pH 3.5-4.0. It was identified as Lactiplantibacillus plantarum by morphological observation and 16S rRNA sequencing analysis. Because strain r1 was isolated from culture ponds and exerted strong inhibitory effects on pathogenic bacteria, it holds potential as an agent to prevent and control infectious diseases in aquaculture. This study provides a foundation for the development and utilization of probiotics in aquaculture.

Reduced graphene oxide increases cells with enlarged outer membrane of Citrifermentans bremense and exopolysaccharides secretion

Conductive carbons can boost anaerobic microbial metabolism by assisting extracellular electron transfer (EET), and their chemistry affects microbial metabolism. Graphene oxide (GO), a chemically oxidized sheet of graphite, has been used in various bioelectrochemical systems, although its mechanism is rarely understood. This study revealed specific metabolic responses to reduced GO (rGO) in an electrogenic strain R4 of Citrifermentans bremense, recently renamed from “Geobacter bremensis,” in comparison to that with graphite felt (GF). Specifically, the change in growth from planktonic cells to biofilm with an enlarged outer membrane. The mRNA profile supported the fact that rGO upregulated the 14 genes related to the exopolysaccharides (EPS) secretion and biofilm formation, which is more than that in GF (4 genes). While GF upregulated the 35 genes involved in cell motility, which is more than that in rGO (8 genes). The heme protein profile suggested that both carbons induced similar EET pathways involving OmcA/MtrC and OmcS; however, GO specifically induced PilQ. These findings show that the chemistry of conductive carbon differentiates metabolism, especially affecting cellular morphology or living form, rather than electron transfer metabolism.

Responses of Mytilus galloprovincialis to challenge with environmental isolates of the potential emerging pathogen Malaciobacter marinus

Bacteria of the Arcobacter-like spp. represent emerging foodborne zoonotic pathogens in humans and animals. Their increasing presence in seafood, suggesting higher occurrence in seawater due to marine pollution, is raising some environmental concern. Although Arcobacter is frequently detected in diseased oysters and stressed bivalve species, no data are available so far on its potential pathogenicity or interactions with the immune system of the bivalve host.In this work, responses to challenge with two strains of Malaciobacter marinus IRTA-19-131 and IRTA-19-132, R1 and R2), isolated from adult Crassostrea gigas during a mortality event in 2019 in Spain, were investigated in the mussel Mytilus galloprovincialis.In vivo experiments were performed in larvae (48 h post-fertilization), and in adult mussels at 24 h post-injection, in order to evaluate the pathogenicity for early developmental stages, and the hemolymph immune responses, respectively. Both R1 and R2 were moderately pathogenic to early larvae, with significant decreases in the development of normal D-veligers from 104 and 103 CFU/mL, respectively. In adults, both strains decreased hemocyte lysosomal membrane stability (LMS), and stimulated extracellular defense responses (ROS production and lysozyme activity).The interactions between mussel hemocytes and M. marinus were investigated in in vitro short-term experiments (30–90 min) using the R1 strain (106–108 CFU/mL). R1 decreased LMS and induced lysosomal enlargement, but not cell detachment or death, and stimulated extracellular ROS production and lysozyme release, confirming in vivo data. Moreover, lysosomal internalization and degradation of bacteria were observed, together with changes in levels of activated mTor and LC3, indicating phagocytic activity. Overall, the results indicate the activation of both extracellular and intracellular immune defenses against M. marinus R1. Accordingly, these responses resulted in a significant hemolymph bactericidal activity, with a large contribution of hemolymph serum.The results represent the first data on the potential pathogenicity of Arcobacter isolated from a shellfish mortality to bivalve larvae and adults, and on their interactions with the immune system of the host.

An automatic fluorescence phenotyping platform to evaluate dynamic infection process of Tobacco mosaic virus-green fluorescent protein in tobacco leaves.

Tobacco is one of the important economic crops all over the world. Tobacco mosaic virus (TMV) seriously affects the yield and quality of tobacco leaves. The expression of TMV in tobacco leaves can be analyzed by detecting green fluorescence-related traits after inoculation with the infectious clone of TMV-GFP (Tobacco mosaic virus - green fluorescent protein). However, traditional methods for detecting TMV-GFP are time-consuming and laborious, and mostly require a lot of manual procedures. In this study, we develop a low-cost machine-vision-based phenotyping platform for the automatic evaluation of fluorescence-related traits in tobacco leaf based on digital camera and image processing. A dynamic monitoring experiment lasting 7 days was conducted to evaluate the efficiency of this platform using Nicotiana tabacum L. with a total of 14 samples, including the wild-type strain SR1 and 4 mutant lines generated by RNA interference technology. As a result, we found that green fluorescence area and brightness generally showed an increasing trend over time, and the trends were different among these SR1 and 4 mutant lines samples, where the maximum and minimum of green fluorescence area and brightness were mutant-4 and mutant-1 respectively. In conclusion, the platform can full-automatically extract fluorescence-related traits with the advantage of low-cost and high accuracy, which could be used in detecting dynamic changes of TMV-GFP in tobacco leaves.

Draft Genome Sequence of Streptomyces sp. Strain R1, Isolated from Water Canal Sediments, Possessing Antimicrobial and Plant Growth Promoting Capabilities.

ABSTRACTWe present the genome sequence of Streptomyces sp. strain R1, isolated from water canal sediments and possessing genes responsible for antimicrobial metabolites and plant growth promotion. The genome assembly contains 7,936,694 bp with 72.24% of guanine-cytosine content. This genome will provide basic knowledge of the genes and pathways involved in the above mechanisms.

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae

PurposeIn Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. Materials and methodsPneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to β-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. ResultsIn PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. ConclusionsNVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.

Characteristics of Vacuolar Inclusions in Coelastrella rubescens Namsu R1 Green Microalgae Cells in Low- and High-Intensity Light

Coelastrella rubescens Kaufnerová & Eliás (Chlorophyceae) is a green, single-celled algae that lives in the terrestrial-air environment. Under stress conditions, its cells go into a state characterized by low photosynthetic activity and high content of reserve lipids and secondary carotenoids. For the first time, a comparative morphological, ultrastructural, and elemental analysis of vacuolar inclusions in the C. rubescens NAMSU R1 strain when cultivated on a mineral medium under conditions of low and high (causing stress) light intensity. Microalgae cells stained with the fluorescent dye DAPI showed signs of the presence of polyphosphates. Polarization microscopy in cells of C. rubescens has identified structures capable of refracting polarized light, which is typical of crystals. Cell analysis of C. rubescens with the transmission electron microscopy (TEM) method revealed the presence of various vacuoles with heterogeneous contents (autophagic bodies, crystalloids, and rounded globules of inhomogeneous electron density). With the exception of autophagic bodies noted in cells only in bright light, these inclusions were characteristic of microalgae cells, regardless of the intensity of illumination. The elemental composition of vacuolar inclusions was characterized by TEM in combination with energy-dispersive X-ray spectroscopy: the predominant content of nitrogen, phosphorus, or both elements simultaneously was established in them. The potential physiological role of C. rubescens vacuolar inclusions is discussed.

Biocontrol Potential of Endophytic Plant-Growth-Promoting Bacteria against Phytopathogenic Viruses: Molecular Interaction with the Host Plant and Comparison with Chitosan.

Endophytic plant-growth-promoting bacteria (ePGPB) are interesting tools for pest management strategies. However, the molecular interactions underlying specific biocontrol effects, particularly against phytopathogenic viruses, remain unexplored. Herein, we investigated the antiviral effects and triggers of induced systemic resistance mediated by four ePGPB (Paraburkholderia fungorum strain R8, Paenibacillus pasadenensis strain R16, Pantoea agglomerans strain 255-7, and Pseudomonas syringae strain 260-02) against four viruses (Cymbidium Ring Spot Virus—CymRSV; Cucumber Mosaic Virus—CMV; Potato Virus X—PVX; and Potato Virus Y—PVY) on Nicotiana benthamiana plants under controlled conditions and compared them with a chitosan-based resistance inducer product. Our studies indicated that ePGPB- and chitosan-treated plants presented well-defined biocontrol efficacy against CymRSV and CMV, unlike PVX and PVY. They exhibited significant reductions in symptom severity while promoting plant height compared to nontreated, virus-infected controls. However, these phenotypic traits showed no association with relative virus quantification. Moreover, the tested defense-related genes (Enhanced Disease Susceptibility-1 (EDS1), Non-expressor of Pathogenesis-related genes-1 (NPR1), and Pathogenesis-related protein-2B (PR2B)) implied the involvement of a salicylic-acid-related defense pathway triggered by EDS1 gene upregulation.

Agronomic Performance of Soybean with Bradyrhizobium Inoculation in Double-Cropped Farming

Land degradation is a serious problem in arid regions, including in Central Asian countries. Soybean symbiosis with rhizobia microbes has an essential role in improving crop productivity and sustaining soil fertility in an arid environment. An experiment was conducted in light straw-colored sierozem soils in the Syrdarya region of Uzbekistan (41.4° N, 64.6° E) under arid conditions over the 2016–2017 and 2017–2018 growing seasons. This study aimed to assess the beneficial N fixation (BNF) ability of soybean in association with the Bradyrhizobium R6 strain and the Bradyrhizobium japonicum USDA110 strains and their combined effect on soil fertility and crop yield. The residues of winter wheat and soybean improved soil structure, i.e., soil humus and N and P contents, significantly differing from those on the soybean followed by summer fallow treatment. Furthermore, soybean in association with dual inoculation had the highest N derived from the atmosphere (Ndfa) (62.9 kg N ha−1), followed by individual soybean treatments with the R6 and USDA110 strains at 51.9 and 40.6 kg N ha−1, respectively. Improved soil quality positively impacted crop output, increasing winter wheat and soybean yields by 36.5% and 34.6%, respectively. Likewise, the yield parameters, i.e., the number of pods, weight of grain per pods, and 1000 seeds were significantly higher in the inoculated treatment with the highest value observed in the dual-inoculated treatment. These results suggest the insertion of soybean with symbiotic bacteria into the cropping system has considerable potential to contribute to sustainable land management practices in arid zones.

ISOLATION, CHARACTERIZATION AND IDENTIFICATION OF AN As(V)-RESISTANT PLANT GROWTH PROMOTING RHIZOBACTERIUM ASSOCIATED WITH THE RHIZOSPHERE OF Azolla microphylla

In the present study, bacterial strains were obtained from the rhizosphere of Azolla microphylla Kaulf. and their arsenic (As) tolerance was tested under in-vitro conditions. Out of the two bacteria screened for As(V) tolerance, strain R1 was found to have the highest As(V) tolerance level at 2000 mg/l and was found to retain up to 58.76% of As given in the medium solution within its cells, as revealed by inductively coupled plasma optical emission spectroscopy analysis. This efficient As(V)-resistant strain was identified using16S rRNA sequencing and fatty acid methyl ester profile analysis, and was found to be a strain of Bacillus cereus (GenBank accession number MH819519). Along with showing abundance of hallmark fatty acids of the Bacillus cereus group, some unique fatty acids were also detected in the bacterial strain R1, namely 15:1 ω5C, 17:1 iso ω10C, 17:1 ante isoA, 17:0, 18:0 iso, 18:1 ω9c, and 17:1 iso ω5C. At its maximum tolerable As(V) dose of 2000 mg/l, strain R1 showed 59.9% As(V) reduction, which was assessed through arsenate reductase enzyme activity. Not only this, under As(V) stress, strain R1 was found to produce 20.4% more cellular protein and display 30.3% antioxidant activity assayed as DPPH reduction. The bacterium is also capable of exhibiting plant growth promoting traits such as solubilisation of inorganic phosphate, production of siderophore and biosynthesis of the plant growth regulator indole acetic acid. Thus, this bacterium has the potential to remediate As(V) from environment and also assist the growth of plants growing in As-polluted ecosystems.

Genome Features and AntiSMASH Analysis of an Endophytic Strain Fusarium sp. R1.

Endophytic fungi are one of the most prolific sources of functional biomolecules with therapeutic potential. Besides playing an important role in serious plant diseases, Fusarium strains possess the powerful capability to produce a diverse array of bioactive secondary metabolites (SMs). In order to in-depth mine gene clusters for SM biosynthesis of the genus Fusarium, an endophytic strain Fusarium sp. R1 isolated from Rumex madaio Makino was extensively investigated by whole-genome sequencing and in-depth bioinformatic analysis, as well as antiSMASH annotation. The results displayed that strain R1 harbors a total of 51.8 Mb genome, which consists of 542 contigs with an N50 scaffold length of 3.21 Mb and 50.4% GC content. Meanwhile, 19,333 functional protein-coding genes, 338 tRNA and 111 rRNA were comprehensively predicted and highly annotated using various BLAST databases including non-redundant (Nr) protein sequence, nucleotide (Nt) sequence, Swiss-Prot, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), as well as Pathogen Host Interactions (PHI) and Carbohydrate-Active enzymes (CAZy) databases. Antibiotics and Secondary Metabolites Analysis Shell (AntiSMASH) results showed that strain R1 has 37 SM biosynthetic gene clusters (BGCs), including 17 nonribosomal peptide synthetases (NRPSs), 13 polyketide synthetases (PKSs), 3 terpene synthases (Ts), 3 hybrid NRPS + PKS and 1 hybrid indole + NRPS. These findings improve our knowledge of the molecular biology of the genus Fusarium and would promote the discovery of new bioactive SMs from strain R1 using gene mining strategies including gene knockout and heteroexpression.

Rhizobacteria of Bali With Obvious Growth-Promoting Properties on Corn (Zea mays L.)

Corn productivity in Indonesia is still relatively low compared with other countries. Therefore, it is necessary to increase the productivity of corn by using rhizobacteria, which have multiple traits. This study was conducted to obtain indigenous rhizobacteria of Bali that have multiple traits, can produce indoleacetic acid (IAA), fix nitrogen from atmosphere, produce siderophores, colonize roots, increase seed germination, and promote the growth of corn. Isolation of rhizobacteria was carried out from the rhizosphere of plants belonging to the Gramineae family that grows in Bali Island, Indonesia. Six isolates, namely, Sr3, Tb9, Rg1, Rg23, Al27, and Jg8, could produce IAA, fix nitrogen from the atmosphere, produce siderophores, and increased germination rate and vigor index of corn seedling. Among them, three isolates, namely, Rg1, Sr3, and Jg8, significantly (p <0.05) increased the germination rate of corn seeds, increased vigor index, increased root dry weight and shoot dry weight of corn at the age of 7 days, and were able to colonize corn roots. Compared with the control, the rhizobacteria treatment increased the germination of corn seeds ranging from 5.04 to 13.05%. Based on the analysis of the 16S rRNA gene, it was found that these rhizobacteria species were Glutamicibacter nicotianae strain Rg1 (accession number OM349119), Brevibacillus invocatus strain Sr3 (accession number OM327515), and Micrococcus luteus strain Jg8 (accession number OM362349). Under a greenhouse condition, all the three isolates significantly (p <0.05) increased nutrient uptake, the leaf chlorophyll content, net assimilation rate, and crop growth rate of corn when compared with control. These results suggested that these isolates of rhizobacteria obviously promoted the growth of corn and can be developed as biostimulant to promote the growth and increase the corn yield in Bali, Indonesia.

Genomic Analyses of Rose Crown Gall-Associated Bacteria Revealed Two New Agrobacterium Species: Agrobacterium burrii sp. nov. and Agrobacterium shirazense sp. nov.

Agrobacterium tumefaciens species complex contains a set of diverse bacterial strains, most of which are well known for their pathogenicity on agricultural plants causing crown gall diseases. Members of A. tumefaciens species complex are classified into several taxonomically distinct lineages called "genomospecies" (13 genomospecies until early 2021). Recently, two genomospecies, G19 (strains RnrT, Rew, and Rnw) and G20 (strains OT33T and R13) infecting Rosa sp. plants in Iran, were described based on biochemical and molecular-phylogenetic data. Whole genome sequence-based core-genome phylogeny followed by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations performed in this study suggested that genomospecies G19 and G20 could be described as two novel and standalone species. In the phylogenetic tree, these two new genomospecies were clustered separately from other genomospecies/species of A. tumefaciens species complex. Moreover, both ANI and dDDH indices between the G19/G20 strains and other Rhizobiaceae members are clearly below the accepted thresholds for prokaryotic species description. Hence, Agrobacterium burrii sp. nov. is proposed to encompass the G19 strains, with RnrT = CFBP 8705T = DSM 112541T as type strain. Agrobacterium shirazense sp. nov. is also proposed to include G20 strains, with OT33T = CFBP 8901T = DSM 112540T as type strain.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Oxytetracycline and Streptomycin Resistance Genes in Xanthomonas arboricola pv. pruni, the Causal Agent of Bacterial Spot in Peach.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot, a major worldwide disease of Prunus species. Very few chemical management options are available for this disease and frequent applications of oxytetracycline (OTC) in the United States peach orchards have raised concerns about resistance development. During 2017–2020, 430 Xap strains were collected from ten peach orchards in South Carolina. Seven OTC-resistant (OTCR) Xap strains were found in 2017 and 2020 from four orchards about 20–270 km apart. Interestingly, the seven strains were also resistant to streptomycin (STR). Six strains grew on media amended with ≤100 μg/mL OTC, while one strain, R1, grew on ≤250 μg/mL OTC. Genome sequence analysis of four representative OTCR strains revealed a 14–20 kb plasmid carrying tetC, tetR, and strAB in each strain. These three genes were transferable to Xanthomonas perforans via conjugation, and they were PCR confirmed in all seven OTCR Xap strains. When tetC and tetR were cloned and expressed together in a sensitive strain, the transconjugants showed resistance to ≤100 μg/mL OTC. When tetC was cloned and expressed alone in a sensitive strain, the transconjugants showed resistance to ≤250 μg/mL OTC. TetC and tetR expression was inducible by OTC in all six wild-type strains resistant to ≤100 μg/mL OTC. However, in the R1 strain resistant to ≤250 μg/mL OTC, tetR was not expressed, possibly due to the presence of Tn3 in the tetR gene, and in this case tetC was constitutively expressed. These data suggest that tetC confers OTC resistance in Xap strains, and tetR regulates the level of OTC resistance conferred by tetC. To our knowledge, this is the first report of OTC resistance in plant pathogenic xanthomonads.