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.

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).

Synergistic enhancement of beta-lactam antibiotics by modified tunicamycin analogs TunR1 and TunR2.

The ß-lactams are the most widely used group of antibiotics in human health and agriculture, but this is under threat due to the persistent rise of pathogenic resistance. Several compounds, including tunicamycin (TUN), can enhance the antibacterial activity of the ß-lactams to the extent of overcoming resistance, but the mammalian toxicity of TUN has precluded its use in this role. Selective hydrogenation of TUN produces modified compounds (TunR1 and TunR2), which retain the enhancement of ß-lactams while having much lower mammalian toxicity. Here we show that TunR1 and TunR2 enhance the antibacterial activity of multiple ß-lactam family members, including penems, cephems, and third-generation penicillins, to a similar extent as does the native TUN. Eleven of the ß-lactams tested were enhanced from 2 to >256-fold against Bacillus subtilis, with comparable results against a penicillin G-resistant strain. The most significant enhancements were obtained with third-generation aminothiazolidyl cephems, including cefotaxime, ceftazidime, and cefquinome. These results support the potential of low toxicity tunicamycin analogs (TunR1 and TunR2) as clinically valid, synergistic enhancers for a broad group of ß-lactam antibiotics.

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.

Haenamindole and fumiquinazoline analogs from a fungicolous isolate of Penicillium lanosum.

Three amino acid-derived compounds, haenamindole (1) and 2'-epi-fumiquinazolines C (2) and D (3), were isolated from cultures of a fungicolous isolate of Penicillium lanosum (MYC-1813=NRRL 66231). Compound 1 was also encountered in cultures of P. corylophilum (MYC-418=NRRL 28126). Structure elucidation of these metabolites was based mainly on high resolution mass spectrometry and NMR data analysis. Haenamindole (1) was found to be a recently reported diketopiperazine-type metabolite that incorporates an unusual ß-Phe unit. Analysis of X-ray crystallographic data and the products of acid hydrolysis of 1 enabled a conclusive, slightly modified stereochemical assignment for haenamindole. Fumiquinazoline analog 2 is a new natural product, while related compound 3 has been previously reported only as a product of an in vitro enzymatic step and of a genetically engineered fungal culture. Compounds 1 and 3 showed antiinsectan activity against the fall armyworm Spodoptera frugiperda.

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.

Draft genome of the most devastating insect pest of coffee worldwide: the coffee berry borer, Hypothenemus hampei.

The coffee berry borer, Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complex polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. The draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies.

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.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

Helicoverpa zea (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Lepidoptera: Noctuidae) Responses to Sorghum bicolor (Poales: Poaceae) Tissues From Lowered Lignin Lines.

The presence of lignin within biomass impedes the production of liquid fuels. Plants with altered lignin content and composition are more amenable to lignocellulosic conversion to ethanol and other biofuels but may be more susceptible to insect damage where lignin is an important resistance factor. However, reduced lignin lines of switchgrasses still retained insect resistance in prior studies. Therefore, we hypothesized that sorghum lines with lowered lignin content will also retain insect resistance. Sorghum excised leaves and stalk pith Sorghum bicolor (L.) Moench (Poales: Poaceae) from near isogenic brown midrib (bmr) 6 and 12 mutants lines, which have lowered lignin content and increased lignocellulosic ethanol conversion efficiency, were examined for insect resistance relative to wild-type (normal BTx623). Greenhouse and growth chamber grown plant tissues were fed to first-instar larvae of corn earworms, Helicoverpa zea (Boddie) and fall armyworms Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), two sorghum major pests. Younger bmr leaves had significantly greater feeding damage in some assays than wild-type leaves, but older bmr6 leaves generally had significantly less damage than wild-type leaves. Caterpillars feeding on the bmr6 leaves often weighed significantly less than those feeding on wild-type leaves, especially in the S. frugiperda assays. Larvae fed the pith from bmr stalks had significantly higher mortality compared with those larvae fed on wild-type pith, which suggested that bmr pith was more toxic. Thus, reducing lignin content or changing subunit composition of bioenergy grasses does not necessarily increase their susceptibility to insects and may result in increased resistance, which would contribute to sustainable production.

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.

Bioactive natural products from fungicolous Hawaiian isolates: secondary metabolites from a Phialemoniopsis sp.

Chemical investigations of two fungal isolates initially identified as members of the genus Phialemonium are described. Both isolates were obtained as colonists of other fungi collected on the island of Hawaii and were later assigned as P. curvatum. However, P. curvatum has recently been reclassified as a member of a new genus (Phialemoniopsis) and renamed as Phialemoniopsis curvata. Studies of solid-substrate fermentation cultures of one of these isolates afforded an oxirapentyn analogue and destruxin A4 as major components, while analysis of the second strain led to the isolation of several simple aromatic metabolites and a compound of mixed biogenetic origin called gabusectin that had previously been reported only in a patent. Structures were assigned mainly by detailed nuclear magnetic resonance and mass spectrometry analysis, and those of two of the major components were confirmed by X-ray crystallography. This report constitutes the first description of secondary metabolites from a member of the genus Phialemoniopsis.

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.

Antimicrobial and antiinsectan phenolic metabolites of Dalea searlsiae.

Continued interest in the chemistry of Dalea spp. led to investigation of Dalea searlsiae, a plant native to areas of the western United States. Methanol extractions of D. searlsiae roots and subsequent chromatographic fractionation afforded the new prenylated and geranylated flavanones malheurans A-D (1-4) and known flavanones (5 and 6). Known rotenoids (7 and 8) and isoflavones (9 and 10) were isolated from aerial portions. Structure determination of pure compounds was accomplished primarily by extensive 1D- and 2D-NMR spectroscopy. The absolute configurations of compounds 1-5, 7, and 8 were assigned using electronic circular dichroism spectroscopy. Antimicrobial bioassays revealed significant activity concentrated in the plant roots. Compounds 1-6 exhibited MICs of 2-8 µg/mL against Streptococcus mutans, Bacillus cereus, and oxacillin-sensitive and -resistant Staphylococcus aureus. Aerial metabolites 7-10 were inactive against these organisms, but the presence of 7 and 8 prompted investigation of the antiinsectan activity of D. searlsiae metabolites toward the major crop pest Spodoptera frugiperda (fall armyworm). While compounds 1-10 all caused significant reductions in larval growth rates, associated mortality (33-66%) was highest with flavanone 4 and rotenoids 7 and 8. These findings suggest a differential allocation of antimicrobial and antiinsectan plant resources to root and aerial portions of the plant, respectively.

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.

Effects of elevated peroxidase levels and corn earworm feeding on gene expression in tomato.

Microarray analysis was used to measure the impact of herbivory by Helicoverpa zea, (corn earworm caterpillar) on wild-type and transgenic tomato, Solanum lycopersicum, plants that over-express peroxidase. Caterpillar herbivory had by far the greatest affect on gene expression, but the peroxidase transgene also altered the expression of a substantial number of tomato genes. Particularly high peroxidase activity resulted in the up-regulation of genes encoding proteinase inhibitors, pathogenesis-related (PR) proteins, as well as proteins associated with iron and calcium transport, and flowering. In a separate experiment conducted under similar conditions, real-time quantitative polymerase chain reaction (qPCR) analysis confirmed our microarray results for many genes. There was some indication that multiple regulatory interactions occurred due to the interaction of the different treatments. While herbivory had the greatest impact on tomato gene expression, our results suggest that levels of expression of a multifunctional gene, such as peroxidase and its products, can influence other gene expression systems distinct from conventional signaling pathways, further indicating the complexity of plant defensive responses to insects.

Enhanced pest resistance of maize leaves expressing monocot crop plant-derived ribosome-inactivating protein and agglutinin.

Although many insect resistance genes have been identified, the number of studies examining their effects in combination using transgenic systems is limited. This study introduced a construct into maize containing the coding sequence for maize ribosome-inactivating protein (MRIP) and wheat germ agglutinin (WGA). Many transformants produced both the MRIP and WGA in leaves. Mature leaves expressing higher levels of these two proteins were more resistant to feeding by first-instar larvae of fall armyworms (Spodoptera frugiperda) and corn earworms (Helicoverpa zea), and the level of resistance was correlated with levels of MRIP and WGA. There was also some indication that resistance to Fusarium verticillioides was increased in the transgenic plant leaves. No statistically significant synergism or antagonism occurred between the activities of the two proteins. MRIP and WGA represent compatible class examples of food plant-derived proteins for multigene resistance to insects.