Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium.

The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.

Potential Biocontrol Agents of Corn Tar Spot Disease Isolated from Overwintered Phyllachora maydis Stromata.

Tar spot disease in corn, caused by Phyllachora maydis, can reduce grain yield by limiting the total photosynthetic area in leaves. Stromata of P. maydis are long-term survival structures that can germinate and release spores in a gelatinous matrix in the spring, which are thought to serve as inoculum in newly planted fields. In this study, overwintered stromata in corn leaves were collected in Central Illinois, surface sterilized, and caged on water agar medium. Fungi and bacteria were collected from the surface of stromata that did not germinate and showed microbial growth. Twenty-two Alternaria isolates and three Cladosporium isolates were collected. Eighteen bacteria, most frequently Pseudomonas and Pantoea species, were also isolated. Spores of Alternaria, Cladosporium, and Gliocladium catenulatum (formulated as a commercial biofungicide) reduced the number of stromata that germinated compared to control untreated stromata. These data suggest that fungi collected from overwintered tar spot stromata can serve as biological control organisms against tar spot disease.

A comparison of transporter gene expression in three species of Peronospora plant pathogens during host infection.

Protein transporters move essential metabolites across membranes in all living organisms. Downy mildew causing plant pathogens are biotrophic oomycetes that transport essential nutrients from their hosts to grow. Little is known about the functions and gene expression levels of membrane transporters produced by downy mildew causing pathogens during infection of their hosts. Approximately 170-190 nonredundant transporter genes were identified in the genomes of Peronospora belbahrii, Peronospora effusa, and Peronospora tabacina, which are specialized pathogens of basil, spinach, and tobacco, respectively. The largest groups of transporter genes in each species belonged to the major facilitator superfamily, mitochondrial carriers (MC), and the drug/metabolite transporter group. Gene expression of putative Peronospora transporters was measured using RNA sequencing data at two time points following inoculation onto leaves of their hosts. There were 16 transporter genes, seven of which were MCs, expressed in each Peronospora species that were among the top 45 most highly expressed transporter genes 5-7 days after inoculation. Gene transcripts encoding the ADP/ATP translocase and the mitochondrial phosphate carrier protein were the most abundant mRNAs detected in each Peronospora species. This study found a number of Peronospora genes that are likely critical for pathogenesis and which might serve as future targets for control of these devastating plant pathogens.

Natural food flavour (E)-2-hexenal, a potential antifungal agent, induces mitochondria-mediated apoptosis in Aspergillus flavus conidia via a ROS-dependent pathway.

Natural food flavour (E)-2-hexenal, a green leaf volatile, exhibits potent antifungal activity on Aspergillus flavus, but its antifungal mechanism has not been fully elucidated. In this study, we evaluated (E)-2-hexenal-induced apoptosis in A. flavus conidia and explored the underlying mechanisms of action. Evidence of apoptosis in A. flavus conidia were investigated by methods including fluorescent staining, flow cytometry, confocal laser scanning microscope, and spectral analysis. Results indicated that 4.0 µL/mL (minimum fungicidal concentration, MFC) of (E)-2-hexenal application induced early markers of apoptotic cell death in A. flavus conidia with a rate of 38.4% after 6 h exposure. Meanwhile, typical hallmarks of apoptosis, such as decreased mitochondrial membrane potential (MMP), activated metacaspase activity, fragmented DNA, mitochondrial permeability transition pore (MPTP) opening and cytochrome c (Cyt C) release from mitochondria to the cytosol were also confirmed. Furthermore, intracellular ATP levels were reduced by 63.3 ± 3.6% and reactive oxygen species (ROS) positive cells increased by 31.1 ± 3.1% during A. flavus apoptosis induced by (E)-2-hexenal. l-Cysteine (Cys), an antioxidant, could strongly block the excess ROS generation caused by (E)-2-hexenal, which correspondingly resulted in a significant inhibition of MPTP opening and decrease of apoptosis in A. flavus, indicating that ROS palys a pivotal role in (E)-2-hexenal-induced apoptosis. These results suggest that (E)-2-hexenal exerts its antifungal effect on A. flavus conidia via a ROS-dependent mitochondrial apoptotic pathway.

Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures.

A Gram-stain positive, aerobic, motile, rod-shaped bacterium designated as strain CBP-2801T was isolated as a contaminant from a culture containing maize callus in Peoria, Illinois, United States. The strain is unique relative to other Cohnella species due to its slow growth and reduced number of sole carbon sources. Phylogenetic analysis using 16S rRNA indicated that strain CBP-2801T is a Cohnella bacterium and showed the highest similarity to Cohnella xylanilytica (96.8%). Genome-based phylogeny and genomic comparisons based on average nucleotide identity confirmed the strain to be a novel species of Cohnella. Growth occurs at 15-45 °C (optimum 40 °C), pH 5-7 (optimum pH 6) and with 0-1% NaCl. The predominant fatty acids are anteiso-15:0 and 18:1 ω6c. Genome mining for secondary metabolites identified a putative biosynthetic cluster that encodes for a novel lasso peptide. In addition, this study contributes five new genome assemblies of type strains of Cohnella species, a genus with less than 30% of the type strains sequenced. The DNA G + C content is 58.7 mol %. Based on the phenotypic, phylogenetic and biochemical data strain CBP-2801T represents a novel species, for which the name Cohnella zeiphila sp. nov. is proposed. The type strain is CBP-2801T (= DSM 111598 = ATCC TSD-230).

Brevibacillus fortis NRS-1210 produces edeines that inhibit the in vitro growth of conidia and chlamydospores of the onion pathogen Fusarium oxysporum f. sp. cepae.

Onions can be damaged by Fusarium basal rot caused by the soilborne fungus Fusarium oxysporum f. sp. cepae (FOC). Control of this pathogen is challenging since there is limited genetic resistance in onion. The identification of molecules that inhibit this pathogen is needed. Antagonism screening showed Brevibacillus fortis NRS-1210 secreted antifungal compounds into growth medium. The spent growth medium, diluted 1:1, inhibited growth of FOC conidia after seven hours and killed 67-91% of conidia after 11 h. The spent medium also inhibited growth of propagules from F. graminearum, F. proliferatum, F. verticillioides and Galactomyces citri-aurantii. Full strength spent growth medium did not effectively kill FOC conidia and chlamydospores inoculated into a sand cornmeal mixture. In silico analysis of the B. fortis NRS-1210 genome indicated the biosynthetic clusters of several antibiotics. Fractionation of spent medium followed by reverse-phase liquid chromatography with tandem mass spectrometry analysis found that fractions with the most antifungal activity contained a combination of edeines A, B and F and no other recognized antibiotics. 1H NMR signals of the active fraction corresponded to edeine, a pentapeptide with broad spectrum antimicrobial activity which blocks translation in both prokaryotes and eukaryotes. Comparative genomics of Brevibacillus genomes shows edeine producers form a clade which consists of: Brevibacillus brevis, Brevibacillus formosus, 'Brevibacillus antibioticus', Brevibacillus schisleri, Brevibacillus fortis, and Brevibacillus porteri. This observation suggests edeine played an important role in the evolution and speciation of the Brevibacillus genus.

Characterization of the infection process by Peronospora belbahrii on basil by scanning electron microscopy.

Basil downy mildew caused by Peronospora belbahrii is a disease of sweet basil (Ocimum basilicum) production worldwide. In this study, sweet basil was grown in plant growth chambers and inoculated with sporangia of P. belbahrii harvested from previously infected plants. Plants were placed in closed, clear plastic bags and leaves harvested over time and observed using scanning electron microscopy. In most cases, sporangia germinated myceliogenically on abaxial and adaxial leaf surfaces as early as three days after inoculation. Germ tubes and the tips of hyphae ramifying on leaf surfaces directly penetrated basil leaves to initiate the infection process. Hyphal growth was not observed to gain entrance to the interior of leaves through stomata, though growth over these openings was observed. Most frequently, seven days after inoculation, one or more sporangiophores grew through stomata to produce new sporangia on both the abaxial and adaxial surfaces of leaves. Macroscopic signs of infection were visible on both sides of leaves approximately ten days after inoculation under the conditions of this study. These results contribute to a better understanding of the infection process and disease onset of P. belbahrii and should help in the development of more effective measures for reducing basil downy mildew.

Phylogenomic analysis of the Brevibacillus brevis clade: a proposal for three new Brevibacillus species, Brevibacillus fortis sp. nov., Brevibacillus porteri sp. nov. and Brevibacillus schisleri sp. nov.

During a screen for antifungal activity of Brevibacillus strains in the Northern Regional Research Laboratory collection we identified two strains with strong activity. Subsequent genomic sequencing and phylogenomic analysis revealed that these strains (NRRL NRS-1210T and NRRL B-41110T) are likely novel species. To confirm their taxonomic placement, we conducted a 16S rRNA phylogenetic analysis and subsequently sequenced the genomes of 10 Brevibacillus type strains with a 16S homology > 97%. Phylogenomic analysis of these type strains and of representative Brevibacillus strains deposited in GenBank also identified several novel clades that should be recognised as novel species. For one of these novel clades, we were able to obtain a publicly available isolate (ATCC 35690T) that could serve as a type strain. The three new species were subjected to a polyphasic characterisation to confirm their taxonomic status. Cells of strains NRRL NRS-1210T, NRRL B-41110T and ATCC 35690T are Gram-staining positive, motile and form tan colonies. All three strains are obligate aerobic mesophiles with a broad pH range for growth. The two most prominent fatty acids of the three strains were identified as iso-C15:0 and anteiso-C15:0. The DNA G+C contents of strains NRRL NRS-1210T, NRRL B-41110T and ATCC 35690T are 47.2 mol%, 47.1 mol% and 47.3 mol%, respectively. Based on these characteristics, three novel species are proposed: Brevibacillus fortis sp. nov. (NRRL NRS-1210T = DSM 9886T = ATCC 51666T), Brevibacillus porteri sp. nov. (NRRL B-41110T = KACC 19693T) and Brevibacillus schisleri sp. nov. (ATCC 35690T = LMG 17055T).

Willingness to Participate in Vaccine-Related Clinical Trials among Older Adults.

The purpose of this study is to understand among a convenience sample of 400 adults aged 60 years of age or older (1) reasons for being willing or unwilling to participate in a vaccine clinical research study and (2) overall perceptions about vaccine clinical research. A cross-sectional study using a sample of older adults residing in the metro-Atlanta area and surrounding neighborhoods was conducted. The study questionnaire contained 37 questions, including questions about socio-demographics and perceptions about clinical trial processes. Statistical analysis was conducted using logistic regression. The adjusted modeling results indicated that sex, distance to research clinic, and being informed about the research findings played a role in the likelihood of an elderly person participating in a vaccine study. Males were more likely to participate in clinical trials as compared to females (OR: 2.486; CI: 1.042⁻5.934). Most participants were willing to travel up to 25 miles from the research clinic. Of the respondents, 45% were unlikely to participate if the results of the current trial are not shared. Improving access to clinical trials in terms of distance traveled and ensuring streamlined processes to inform participants about the results of the trial in the future would increase willingness to participate in vaccine clinical trials. The survey could serve as a useful tool for conducting vaccine studies and other clinical trials by understanding the barriers specific to the elderly.

Reducing production of fumonisin mycotoxins in Fusarium verticillioides by RNA interference.

The fungus Fusarium verticillioides is a maize pathogen that can produce fumonisin mycotoxins in ears under certain environmental conditions. Because fumonisins pose health risks to humans and livestock, control strategies with minimal risk to the environment are needed to reduce fumonisin contamination. Host-induced gene silencing is a promising technique in which double-stranded RNA expressed in the plant host is absorbed by an invading fungus and down-regulates genes critical for pathogenicity or mycotoxin production in the fungus. A key preliminary step of this technique is identification of DNA segments within the targeted fungal gene that can effectively silence the gene. Here, we used segments of the fumonisin biosynthetic gene FUM1 to generate double-stranded RNA in F. verticillioides. Several of the resulting transformants exhibited reduced FUM1 gene expression and fumonisin production (24- to 3675-fold reduction in fumonisin FB1). Similar reductions in fumonisin production resulted from double-stranded RNA constructs with segments of FUM8, another fumonisin biosynthetic gene (3.5- to 2240-fold reduction in fumonisin FB1). FUM1 or FUM8 silencing constructs were transformed into three isolates of F. verticillioides. Whole genome sequence analysis of seven transformants revealed that reductions in fumonisin production were not due to mutation of the fumonisin biosynthetic gene cluster and revealed a complex pattern of plasmid integration. These results suggest the cloned FUM1 or FUM8 gene segments could be expressed in maize for host-induced gene silencing of fumonisin production.

A quantitative method for determining relative colonization rates of maize callus by Fusarium graminearum for resistance gene evaluations.

A quantitative PCR method was developed for detecting Fusarium graminearum growing in maize callus. Fungal DNA was detected 12h after inoculation (detection limit, 0.2pg) and was correlated with visual ratings. The method effectively quantified fungal growth in callus overexpressing a peroxidase gene conferring fungal resistance.

Vast potential for using the piggyBac transposon to engineer transgenic plants at specific genomic locations.

The acceptance of bioengineered plants by some nations is hampered by a number of factors, including the random insertion of a transgene into the host genome. Emerging technologies, such as site-specific nucleases, are enabling plant scientists to promote recombination or mutations at specific plant loci. Off target activity of these nucleases may limit widespread use. Insertion of transgenes by transposases engineered with a specific DNA binding domain has been accomplished in a number of organisms, but not in plants. The piggyBac transposon system, originally isolated from an insect, has been utilized to transform a variety of organisms. The piggyBac transposase is amendable to structural modifications, and was able to insert a transgene at a specific human locus through fusion of a DNA binding domain to its N-terminus. Recent developments demonstrating the activity of piggyBac transposase in plants is an important first step toward the potential use of engineered versions of piggyBac transposase for site-specific transgene insertion in plants.

Maize peroxidase Px5 has a highly conserved sequence in inbreds resistant to mycotoxin producing fungi which enhances fungal and insect resistance.

Mycotoxin presence in maize causes health and economic issues for humans and animals. Although many studies have investigated expression differences of genes putatively governing resistance to producing fungi, few have confirmed a resistance role, or examined putative resistance gene structure in more than a couple of inbreds. The pericarp expression of maize Px5 has previously been associated with resistance to Aspergillus flavus growth and insects in a set of inbreds. Genes from 14 different inbreds that included ones with resistance and susceptibility to A. flavus, Fusarium proliferatum, F. verticillioides and F. graminearum and/or mycotoxin production were cloned using high fidelity enzymes, and sequenced. The sequence of Px5 from all resistant inbreds was identical, except for a single base change in two inbreds, only one of which affected the amino acid sequence. Conversely, the Px5 sequence from several susceptible inbreds had several base variations, some of which affected amino acid sequence that would potentially alter secondary structure, and thus enzyme function. The sequence of the maize peroxidase Px5 common to inbreds resistant to mycotoxigenic fungi was overexpressed in maize callus. Callus transformants overexpressing the gene caused significant reductions in growth for fall armyworms, corn earworms, and F. graminearum compared to transformant callus with a ß-glucuronidase gene. This study demonstrates rarer transcripts of potential resistance genes overlooked by expression screens can be identified by sequence comparisons. A role in pest resistance can be verified by callus expression of the candidate genes, which can thereby justify larger scale transformation and regeneration of transgenic plants expressing the resistance gene for further evaluation.

Antifungal Activity of a Synthetic Cationic Peptide against the Plant Pathogens Colletotrichum graminicola and Three Fusarium Species.

A small cationic peptide (JH8944) was tested for activity against a number of pathogens of agricultural crops. JH8944 inhibited conidium growth in most of the tested plant pathogens with a dose of 50 µg/ml, although one isolate of Fusarium oxysporum was inhibited at 5 µg/ml of JH8944. Most conidia of Fusarium graminearum were killed within 6 hours of treatment with 50 µg/ml of JH8944. Germinating F. graminearum conidia required 238 µg/ml of JH8944 for 90% growth inhibition. The peptide did not cause any damage to tissues surrounding maize leaf punctures when tested at a higher concentration of 250 µg/ml even after 3 days. Liposomes consisting of phosphatidylglycerol were susceptible to leakage after treatment with 25 and 50 µg/ml of JH8944. These experiments suggest this peptide destroys fungal membrane integrity and could be utilized for control of crop fungal pathogens.

Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

Environmental effects on resistance gene expression in milk stage popcorn kernels and associations with mycotoxin production.

Like other forms of maize, popcorn is subject to increased levels of contamination by a variety of different mycotoxins under stress conditions, although levels generally are less than dent maize under comparable stress. Gene array analysis was used to determine expression differences of disease resistance-associated genes in milk stage kernels from commercial popcorn fields over 3 years. Relatively lower expression of resistance gene types was noted in years with higher temperatures and lower rainfall, which was consistent with prior results for many previously identified resistance response-associated genes. The lower rates of expression occurred for genes such as chitinases, protease inhibitors, and peroxidases; enzymes involved in the synthesis of cell wall barriers and secondary metabolites; and regulatory proteins. However, expression of several specific resistance genes previously associated with mycotoxins, such as aflatoxin in dent maize, was not affected. Insect damage altered the spectrum of resistance gene expression differences compared to undamaged ears. Correlation analyses showed expression differences of some previously reported resistance genes that were highly associated with mycotoxin levels and included glucanases, protease inhibitors, peroxidases, and thionins.

Identification of a bioactive Bowman-Birk inhibitor from an insect-resistant early maize inbred.

Breeding of maize, Zea mays, has improved insect resistance, but the genetic and biochemical basis of many of these improvements is unknown. Maize oligonucleotide microarrays were utilized to identify differentially expressed genes in leaves of three maize inbreds, parents Oh40B and W8 and progeny Oh43, developed in the 1940s. Oh43 had enhanced leaf resistance to corn earworm larvae, Helicoverpa zea, and fall armyworm larvae, Spodoptera frugiperda, compared to one or both parents. Among ca. 100 significantly differentially expressed genes, expression of a Bowman-Birk trypsin inhibitor (BBI) gene was at least ca. 8-fold higher in Oh43 than in either parent. The Oh43 BBI gene was expressed as a recombinant protein. Purified BBI inhibited trypsin and the growth of fall armyworm larvae when added to insect diet. These experiments indicate that comparative gene expression analysis combined with insect resistance measurements of early inbreds can identify previously unrecognized resistance genes.

A non-autonomous insect piggyBac transposable element is mobile in tobacco.

The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid only requires expression of piggyBac transposase. To determine if piggyBac could function in dicotyledonous plants, a two-element system was developed in tobacco (Nicotiana tabacum) to test for transposable element excision and insertion. The first transgenic line constitutively expressed piggyBac transposase, while the second transgenic line contained at least two non-autonomous piggyBac transposable elements. Progeny from crosses of the two transgenic lines was analyzed for piggyBac excision and transposition. Several progeny displayed excision events, and all the sequenced excision sites exhibited evidence of the precise excision mechanism characteristic of piggyBac transposase. Two unique transposition insertion events were identified that each included diagnostic duplication of the target site. These data indicate that piggyBac transposase is active in a dicotyledonous plant, although at a low frequency.

Expression of a wolf spider toxin in tobacco inhibits the growth of microbes and insects.

Lycotoxin I, from the wolf spider (Lycosa carolinensis), is an amphipathic pore-forming peptide that has antimicrobial and anti-insect activity. Constitutive expression of a lycotoxin I modified for oral toxicity to insects in tobacco (Nicotiana tabacum) conferred significantly enhanced resistance to larvae of the corn earworm (Helicoverpa zea) and cigarette beetle (Lasioderma serricorne). Gene expression levels of modified lycotoxin I were negatively correlated to the survival of corn earworm larvae. In addition, pathogenic symptoms caused by Pseudomonas syringae pathovar tabaci and Alternaria alternata on the modified lycotoxin I-expressing leaves were significantly less severe than on wild type leaves. These results indicate that modified lycotoxin I expression in tobacco can potentially protect leaf tissue from a broad spectrum of pests and pathogens.