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Aphids are a large group of hemipteran pests that affect the physiology, growth, and development of plants by using piercing mouthparts to consume fluids from the host. Based an recent data, aphids modulate the microbiomes of plants and thereby affect the overall outcome of the biological interaction. However, in a few reports, aboveground aphids manipulate the metabolism of the host and facilitate infestations by rhizosphere bacteria (rhizobacteria). In this study, we evaluated whether aphids alter the plant resistance that is mediated by the bacterial community of the root system. The rhizobacteria were affected by aphid infestation of pepper, and a large population of gram-positive bacteria was detected. Notably, Paenibacillus spp. were the unique gram-positive bacteria to respond to changes induced by the aphids. Paenibacillus polymyxa E681 was used as a rhizobacterium model to assess the recruitment of bacteria to the rhizosphere by the phloem-sucking of aphids and to test the effect of P. polymyxa on the susceptibility of plants to aphids. The root exudates secreted from peppers infested with aphids increased the growth rate of P. polymyxa E681. The application of P. polymyxa E681 to pepper roots promoted the colonization of aphids within 2 days of inoculation. Collectively, our results suggest that aphid infestation modulated the root exudation, which led to the recruitment of rhizobacteria that manipulated the resistance of peppers to aphids. In this study, new information is provided on how the infestation of insects is facilitated through insect-derived modulation of plant resistance with the attraction of gram-positive rhizobacteria.

Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.

PacBio's long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of secondgeneration, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

Pseudomonas syringae pv. syringae (Psy) is a major bacterial pathogen of many economically important plant species. Despite the severity of its impact, the genome sequence of the type strain has not been reported. Here, we present the draft genome sequence of Psy ATCC 19310. Comparative genomic analysis revealed that Psy ATCC 19310 is closely related to Psy B728a. However, only a few type III effectors, which are key virulence factors, are shared by the two strains, indicating the possibility of host-pathogen specificity and genome dynamics, even under the pathovar level.

Twenty-nine P. polymyxa strains isolated from rhizospheres of various crops were clustered into five genotypic groups on the basis of BOX-PCR analysis. The characteristics of several plant growth-promoting factors among the isolates revealed the distinct attributes in each allocated group. Under gnotobiotic conditions, inoculation of pepper roots with P. polymyxa isolates significantly increased the biomass in 17 of total 29 treated plants with untreated plants. Experiments on induced systemic resistance (ISR) against bacterial spot pathogen Xanthomonas axonopodis pv. vesicatoria in pepper by P. polymyxa strains were conducted and only one isolate (KNUC265) was selected. Further studies into ISR mediation by the KNUC265 strain against the soft-rot pathogen Erwinia carotovora subsp. carotovora in tobacco demonstrated that the tobacco seedlings exposed to either bacterial volatiles or diffusible metabolites exhibited a reduction in disease severity. In conclusion, ISR and plant growth promotion triggered by P. polymyxa isolates were systemically investigated on pepper for the first time. The P. polymyxa KNUC265 strain, which elicited both ISR and plant growth promotion, could be potentially used in improving the yield of pepper and possibly of other crops.

Bacteria sense their population density and coordinate the expression of target genes, including virulence factors in Gram-negative bacteria, by the N-acylhomoserine lactones (AHLs)-dependent quorum-sensing (QS) mechanism. In contrast, several soil bacteria are able to interfere with QS by enzymatic degradation of AHLs, referred to as quorum quenching. A potent AHL-degrading enzyme, AiiA, of Bacillus thuringiensis has been reported to effectively attenuate the virulence of bacteria by quorum quenching. However, little is known about the role of AiiA in B. thuringiensis itself. In the present study, an aiiA-defective mutant was generated to investigate the role of AiiA in rhizosphere competence in the root system of pepper. The aiiA mutant showed no detectable AHL-degrading activity and was less effective for suppression of soft-rot symptom caused by Erwinia carotovora on the potato slice. On the pepper root, the survival rate of the aiiA mutant significantly decreased over time compared with that of wild type. Interestingly, viable cell count analysis revealed that the bacterial number and composition of E. carotovora were not different between treatments of wild type and the aiiA mutant, although root application of the aiiA mutant in pepper failed to protect the plant from root rot. These results provide evidence that AiiA can play an important role in rhizosphere competentce of B. thuringiensis and bacterial quorum quenching to Gram-negative bacteria without changing bacterial number or composition.

Indole-3-acetic acid (IAA) is produced commonly by plants and many bacteria, however, little is known about the genetic basis involving the key enzymes of IAA biosynthetic pathways from Bacillus spp. IAA intermediates from the Gram-positive spore-forming bacterium Paenibacillus polymyxa E681 were investigated, which showed the existence of only an indole-3-pyruvic acid (IPA) pathway for IAA biosynthesis from the bacterium. Four open reading frames (ORFs) encoding indole-3-pyruvate decarboxylaselike proteins and putative indole-3-pyruvate decarboxylase (IPDC), a key enzyme in the IPA synthetic pathway, were found on the genome sequence database of P. polymyxa and cloned in Escherichia coli DH5alpha. One of the ORFs, PP2_01257, was assigned as probable indole-3-pyruvate decarboxylase. The ORF consisted of 1,743 nucleotides encoding 581 amino acids with a deduced molecular mass of 63,380 Da. Alignment studies of the deduced amino acid sequence of the ORF with known IPDC sequences revealed conservation of several amino acids in PP2_01257, essential for substrate and cofactor binding. Recombinant protein, gene product of the ORF PP2_01257 from P. polymyxa E681, was expressed in E. coli BL21 (DE3) as a glutathione S-transferase (GST)-fusion protein and purified to homogeneity using affinity chromatography. The molecular mass of the purified enzyme showed about 63 kDa, corresponding closely to the expected molecular mass of IPDC. The indole-3-pyruvate decarboxylase activity of the recombinant protein, detected by HPLC, using IPA substrate in the enzyme reaction confirmed the identity and functionality of the enzyme IPDC from the E681 strain.

The role of exopolysaccharides (EPSs) from a plant growth-promoting rhizobacterium, Burkholderia gladioli IN26, on elicitation of induced systemic resistance was investigated. A purified EPS induced expression of PR- 1a::GUS on tobacco and elicited induced resistance against Colletotrichum orbiculare on cucumber. The maximum level of disease protection was noted when seeds were soaked in 200 ppm of the EPS. Our results indicate that EPS from specific rhizobacteria can elicit induced resistance and suggest that bacterial EPS might be a useful elicitor of resistance under field conditions.

Paenibacillus polymyxa E681 is known to be able to suppress plant diseases by producing antimicrobial compounds and to promote plant growth by producing phytohormones, and secreting diverse degrading enzymes. In spite of these capabilities, little is known regarding the flow of information from the bacterial strain to the barley roots. In an attempt to determine the flow of information from the bacterial strain to barley roots, the train was grown in the presence and absence of barley, and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry were used. 2D-PAGE detected approximately 1000 spots in the cell and 1100 spots in the supernatant at a pH 4-10 gradient. Interestingly, about 80 spots from each sample showed quantitative variations. Fifty-three spots from these were analyzed by MALDI-TOF mass spectrometry and 28 proteins were identified. Most of the cytosolic proteins expressed at higher levels were found in P. polymyxa E681 cells grown in the presence of barley rather than in the absence of barley. Proteins detected at a lower level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley were lipoprotein, glucose-6-phosphate 1-dehydrogenase, heat-shock protein HtpG spermidine synthase, OrfZ, ribonuclease PH, and coenzyme PQQ synthesis protein, and flagellar hook-associated protein 2 whereas proteins detected at a higher level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley included D-alanyl-D-alanine ligase A, isopentenyldiphosphate delta-isomerase, ABC transporter ATP-binding protein Uup, lipase. Many of the proteins belonging to plant-induced stimulons are associated with biosynthetic metabolism and metabolites of proteins and transport. Some of these proteins would be expected to be induced by environmental changes resulting from the accumulation of plant-secreted substances.

We evaluated a commercial biopreparation of plant growth-promoting rhizobacteria (PGPR) strains Bacillus subtilis GB03 and B. amyloliquefaciens IN937a formulated with the carrier chitosan (BioYield) for its capacity to elicit growth promotion and induced systemic resistance against infection by Cucumber Mosaic Virus (CMV) and Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana. The biopreparation promoted plant growth of Arabidopsis hormonal mutants, which included auxin, gibberellic acid, ethylene, jasmonate, salicylic acid, and brassinosteroid insensitive lines as well as each wild-type. The biopreparation protected plants against CMV based on disease severity in wild-type plants. However, virus titre was not lower in control plants and those treated with biopreparation, suggesting that the biopreparation induced tolerance rather than resistance against CMV. Interestingly, the biopreparation induced resistance against CMV in NahG plants, as evidenced by both reduced disease severity and virus titer. The biopreparation also elicited induced resistance against P. syringae pv. tomato in the wild-type but not in NahG transgenic plants, which degrade endogenous salicylic acid, indicating the involvement of salicylic acid signaling. Our results indicate that some PGPR strains can elicit plant growth promotion by mechanisms that are different from known hormonal signaling pathways. In addition, the mechanism for elicitation of induced resistance by PGPR may be pathogen-dependent. Collectively, the two-Bacilli strain mixture can be utilized as a biological inoculant for both protection of plant against bacterial and viral pathogens and enhancement of plant growth.

Plant growth-promoting rhizobacteria (PGPR) have the potential to be used as microbial inoculants to reduce disease incidence and severity and to increase crop yield. Some of the PGPR have been reported to be able to enter plant tissues and establish endophytic populations. Here, we demonstrated an approach to screen bacterial endophytes that have the capacity to promote the growth of pepper seedlings and protect pepper plants against a bacterial pathogen. Initially, out of 150 bacterial isolates collected from healthy stems of peppers cultivated in the Chungcheong and Gyeongsang provinces of Korea, 23 putative endophytic isolates that were considered to be predominating and representative of each pepper sample were selected. By phenotypic characterization and partial 16S rDNA sequence analysis, the isolates were identified as species of Ochrobacterium, Pantoea, Pseudomonas, Sphingomonas, Janthinobacterium, Ralstonia, Arthrobacter, Clavibacter, Sporosarcina, Acidovorax, and Brevundimonas. Among them, two isolates, PS4 and PS27, were selected because they showed consistent colonizing capacity in pepper stems at the levels of 10(6)-10(7) CFU/g tissue, and were found to be most closely related to Pseudomonas rhodesiae and Pantoea ananatis, respectively, by additional analyses of their entire 16S rDNA sequences. Drenching application of the two strains on the pepper seedlings promoted significant growth of peppers, enhancing their root fresh weight by 73.9% and 41.5%, respectively. The two strains also elicited induced systemic resistance of plants against Xanthomonas axonopodis pv. vesicatoria.