Tuning the thermostability of GHG gels by salts at different positions on the Hofmeister scale.

The influence of Hofmeister cations (NH4+, Na+, Mg2+) and anions (H2PO4-, CH3COO-, Cl-, NO3-) on the thermostability of a GHG hydrogel was investigated. The combined results of UV circular dichroism (UVCD) and Small Amplitude Oscillatory Shear Rheology experiments reveal that the addition of salt reduces the stability of the gel phase and the underlying fibrils. In line with the cationic Hofmeister hierarchy, the chaotropic Mg2+ ions caused the greatest thermal destabilization of the gel phase with the gel → sol transition temperature Tgs value lowered by 10 °C. In the absence of salt, the gel → sol transition probed by the storage modulus and microscopy is biphasic. In the presence of salt, it becomes monophasic. Contrary to expectations the presence of Hofmeister anions leads to a nearly identical reduction of the gel → sol transition temperatures. However, UVCD spectra suggest that they affect the ππ-stacking between imidazole groups to a different extent. We relate the absence of ion specificity regarding the solubility of fibrils (probed by UVCD) to the observed enthalpy-entropy compensation of the dissolution process. Our results combined show how CD spectroscopy and rheology combined yields a more nuanced picture of the processes underlying the gel → sol transition.

Conformational Manifold Sampled by Two Short Linear Motif Segments Probed by Circular Dichroism, Vibrational, and Nuclear Magnetic Resonance Spectroscopy.

Disordered protein segments called short linear motifs (SLiM) serve as recognition sites for a variety of biological processes and act as targeting signals, modification, and ligand binding sites. While SLiMs do not adopt one of the known regular secondary structures, the conformational distribution might still reflect the structural propensities of their amino acid residues and possible interactions between them. In the past, conformational analyses of short peptides provided compelling evidence for the notion that individual residues are less conformationally flexible than locally expected for a random coil. Here, we combined various spectroscopies (NMR, IR, vibrational, and UV circular dichroism) to determine the Ramachandran plots of two SLiM motifs, i.e., GRRDSG and GRRTSG. They are two representatives of RxxS motifs that are capable of being phosphorylated by protein kinase A, an enzyme that plays a fundamental role in a variety of biological processes. Our results reveal that the nearest and non-nearest interactions between residues cause redistributions between polyproline II and ß-strand basins while concomitantly stabilizing extended relative to turn-forming and helical structures. They also cause shifts in basin positions. With increasing temperature, ß-strand populations become more populated at the expense of polyproline II. While molecular dynamics simulations with Amber ff14SB and CHARMM 36m force fields indicate residue-residue interactions, they do not account for the observed structural changes.

Influence of central sidechain on self-assembly of glycine-x-glycine peptides.

Low molecular weight gelators (LMWGs) are the subject of intense research for a range of biomedical and engineering applications. Peptides are a special class of LMWG, which offer infinite sequence possibilities and, therefore, engineered properties. This work examines the propensity of the GxG peptide family, where x denotes a guest residue, to self-assemble into fibril networks via changes in pH and ethanol concentration. These triggers for gelation are motivated by recent work on GHG and GAG, which unexpectedly self-assemble into centimeter long fibril networks with unique rheological properties. The propensity of GxG peptides to self-assemble, and the physical and chemical properties of the self-assembled structures are characterized by microscopy, spectroscopy, rheology, and X-ray diffraction. Interestingly, we show that the number, length, size, and morphology of the crystalline self-assembled aggregates depend significantly on the x-residue chemistry and the solution conditions, i.e. pH, temperature, peptide concentration, etc. The different x-residues allow us to probe the importance of different peptide interactions, e.g. π-π stacking, hydrogen bonding, and hydrophobicity, on the formation of fibrils. We conclude that fibril formation requires π-π stacking interactions in pure water, while hydrogen bonding can form fibrils in the presence of ethanol-water solutions. These results validate and support theoretical arguments on the propensity for self-assembly and leads to a better understanding of the relationship between peptide chemistry and fibril self-assembly. Overall, GxG peptides constitute a unique family of peptides, whose characterization will aid in advancing our understanding of self-assembly driving forces for fibril formation in peptide systems.

Forbidden Secondary Structures Found in Gel-Forming Fibrils of Glycylphenylalanylglycine.

The zwitterionic l-tripeptide glycylphenylalanylglycine self-assembles into very long crystalline fibrils in an aqueous solution, which causes the formation of an exceptionally strong gel phase (G' ∼ 5 × 106 Pa). The Rietveld refinement analysis of its powder X-ray diffraction (PXRD) pattern reveals a unit cell with four peptides forming a P212121 space group and adopting an inverse polyproline II conformation, that is, a right-handed helical structure that occupies the "forbidden" region of the Ramachandran plot. This unusual structure is stabilized by a plethora of intermolecular interactions facilitated by the large number of different functional groups of the unblocked tripeptide. Comparisons of simulated and experimental Fourier transform infrared and vibrational circular dichroism (VCD) amide I' profiles corroborate the PXRD structure. Our experimental setup reduces the sample to a quasi-two-dimensional network of fibrils. We exploited the influence of this reduced dimensionality on the amide I VCD to identify the main fibril axis. We demonstrate that PXRD, vibrational spectroscopy, and amide I simulations provide a powerful toolset for secondary structure and fibril axis determination.

Repeating Aspartic Acid Residues Prefer Turn-like Conformations in the Unfolded State: Implications for Early Protein Folding.

Protein folding can be described as a motion of the polypeptide chain in a potential energy funnel, where the conformational manifold is narrowed as the chain traverses from a completely unfolded state until it reaches the folded (native) state. The initial folding stages set the tone for this process by substantially narrowing the manifold of accessible conformations. In an ideally unfolded state with no long-range stabilizing forces, local conformations (i.e., residual structures) are likely to drive the folding process. While most amino acid residues tend to predominantly adopt extended structures in unfolded proteins and peptides, aspartic acid exhibits a relatively high intrinsic preference for turn-forming conformations. Regions in an unfolded polypeptide or protein that are rich in aspartic acid residues may therefore be crucial sites for protein folding steps. By combining NMR and vibrational spectroscopies, we observed that the conformational sampling of multiple sequentially neighbored aspartic acid residues in the model peptides GDDG and GDDDG even show an on average higher propensity for turn-forming structures than the intrinsic reference system D in GDG, which suggests that nearest neighbor interactions between adjacent aspartic acid residues stabilize local turn-forming structures. In the presence of the unlike neighbor phenylalanine, nearest neighbor interactions are of a totally different nature in that it they decrease the turn-forming propensities and mutually increase the sampling of polyproline II (pPII) conformations. We hypothesize the structural role of aspartic residues in intrinsically disordered proteins in general, and particularly in small linear motifs, that are very much determined by their respective neighbors.

Stemphylium Leaf Spot of Parsley in California Caused by Stemphylium vesicarium.

From 2009 through 2011, a previously undescribed disease occurred on commercial parsley in coastal (Ventura County) California. Symptoms of the disease consisted of circular to oval, tan to brown leaf spots and resulted in loss of crop quality and, hence, reduced yields. A fungus was consistently isolated from symptomatic parsley. Morphological and molecular data identified the fungus as Stemphylium vesicarium. When inoculated onto parsley leaves, the isolates caused symptoms that were identical to those seen in the field; the same fungus was recovered from test plants, thus completing Koch's postulates. Additional inoculation experiments demonstrated that 10 of 11 tested flat leaf and curly parsley cultivars were susceptible. The parsley isolates also caused small leaf spots on other Apiaceae family plants (carrot and celery) but not on leek, onion, spinach, and tomato. Isolates caused brown lesions to form when inoculated onto pear fruit but only when the fruit tissue was wounded. Using a freeze-blotter seedborne pathogen assay, parsley seed was found to have a low incidence (0.25%) of S. vesicarium. When inoculated onto parsley leaves, three of four isolates from seed caused the same leaf spot disease. This is the first documentation of a foliar parsley disease caused by S. vesicarium. The occurrence of S. vesicarium on parsley seed indicates that infested seed may be one source of initial inoculum. Based on the negative results in the host range experiments, it appears that this parsley pathogen differs from the S. vesicarium that causes disease on leek, garlic, onion, and pear fruit.

A revision of the genus Lomaantha, with the description of a new species.

Lomaantha phragmitis sp. nov. is described and illustrated from a specimen collected on dead culms of Phragmites communis in southern China. The fungus differs from other described Lomaantha species in its conidiophores, conidiogenous cells and conidial appendages. Conidial morphology and presence or absence of percurrent proliferation of conidiogenous cells are the main characters distinguishing species within this genus. We provided a key and synoptic table of morphological characters of all three Lomaantha species.

Elevated atmospheric carbon dioxide concentrations amplify Alternaria alternata sporulation and total antigen production.

BACKGROUND: Although the effect of elevated carbon dioxide (CO2) concentration on pollen production has been established in some plant species, impacts on fungal sporulation and antigen production have not been elucidated. OBJECTIVE: Our purpose was to examine the effects of rising atmospheric CO2 concentrations on the quantity and quality of fungal spores produced on timothy (Phleum pratense) leaves. METHODS: Timothy plants were grown at four CO2 concentrations (300, 400, 500, and 600 micromol/mol). Leaves were used as growth substrate for Alternaria alternata and Cladosporium phlei. The spore abundance produced by both fungi, as well as the size (microscopy) and antigenic protein content (ELISA) of A. alternata, were quantified. RESULTS: Leaf carbon-to-nitrogen ratio was greater at 500 and 600 micromol/mol, and leaf biomass was greater at 600 micromol/mol than at the lower CO2 concentrations. Leaf carbon-to-nitrogen ratio was positively correlated with A. alternata spore production per gram of leaf but negatively correlated with antigenic protein content per spore. At 500 and 600 micromol/mol CO2 concentrations, A. alternata produced nearly three times the number of spores and more than twice the total antigenic protein per plant than at lower concentrations. C. phlei spore production was positively correlated with leaf carbon-to-nitrogen ratio, but overall spore production was much lower than in A. alternata, and total per-plant production did not vary among CO2 concentrations. CONCLUSIONS: Elevated CO2 concentrations often increase plant leaf biomass and carbon-to-nitrogen ratio. Here we demonstrate for the first time that these leaf changes are associated with increased spore production by A. alternata, a ubiquitous allergenic fungus. This response may contribute to the increasing prevalence of allergies and asthma.

Ulocladium cantlous sp. nov. isolated from northwestern China: its morphology and molecular phylogenetic position.

A new species of Ulocladium was isolated from diseased leaves from two Cucumis sp. growing in Sinkiang and Gansu provinces of China. Conidia were isolated from necrotic leaves and used to establish single-spore pure cultures. Conidia were harvested from cultures 7 d after incubation for morphological comparisons. The morphology of this species resembles that of U. botrytis and U. consortiale. However it is distinguished from these two species by the sizes of obovoid to broadly ellipsoidal conidia and longer conidiophores. A taxonomic description of U. cantlous, comparison with related species in this genus, and a species phylogeny based on the partial nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and the Alternaria alternata major allergen (Alt a 1) gene are provided.

Functional analysis of Pcipg2 from the straminopilous plant pathogen Phytophthora capsici.

Phytophthora capsici causes serious diseases in numerous crop plants. Polygalacturonases (PGs) are cell wall-degrading enzymes that play an important role in pathogenesis in straminopilous pathogens. To understand PGs as they relate to the virulence of P. capsici, Pcipg2 was identified from a genomic library of a highly virulent P. capsici strain. Pcipg2 was strongly expressed during symptom development after the inoculation of pepper leaves with P. capsici. The wild protein (PCIPGII) was obtained from the expression of pcipg2 and found that increasing activity of PGs in PCIPGII-treated pepper leaves was consistent with increasing symptom development. Asp residues in active sites within pcipg2 affected PCIPGII activity or its virulence on pepper leaves. Results show that pcipg2 is an important gene among pcipg genes, and illustrate the benefit of analyzing mechanisms of pathogenicity during the period of host/parasite interaction.

Genetic Diversity Among Ophiosphaerella agrostis Strains Causing Dead Spot in Creeping Bentgrass.

Dead spot (Ophiosphaerella agrostis) is a relatively new disease of young creeping bentgrass and hybrid bermudagrass putting greens in the United States. Little is known about the biology or genetic diversity of the pathogen. O. agrostis is unusual in that it produces prodigious numbers of pseudothecia in the field throughout the summer months and has no known asexual state. A total of 77 O. agrostis isolates were collected from 21 different bentgrass putting greens and one hybrid bermudagrass green in 11 states. DNA fingerprint analysis revealed that 78 out of 97 markers were polymorphic (80.4%), providing 57 unique profiles. Genetic variation of O. agrostis was diverse, and isolates separated into three distinct clades with ≥69% similarity. Analysis of molecular variance indicated that the geographic origins of the isolates and the ability to produce pseudothecia were the best indicators for genetic similarity among O. agrostis isolates. Colony color varied among the isolates, but generally was similar for isolates residing within two clades (B and C). Colony color of isolates within clade A appeared to be a mixture of the colony colors exhibited by clades B and C. Isolates examined within each clade generally had varying levels of pseudothecia production and varying colony colors when grown on PDA. Although O. agrostis is a homothallic species, it is unclear if outcrossing among strains occurs.

Environmental Influences on the Release of Ophiosphaerella agrostis Ascospores Under Controlled and Field Conditions.

ABSTRACT Ophiosphaerella agrostis, the causal agent of dead spot of creeping bentgrass (Agrostis stolonifera), can produce prodigious numbers of pseudothecia and ascospores throughout the summer. The environmental conditions and seasonal timings associated with O. agrostis ascospore release are unknown. The objectives of this research were to (i) determine the influence of light and relative humidity on ascospore release in a controlled environment, (ii) document the seasonal and daily discharge patterns of ascospores in the field, and (iii) elucidate environmental conditions that promote ascospore release under field conditions. In a growth chamber, a sharp decrease (100 to approximately 50%; 25 degrees C) in relative humidity resulted in a rapid (1- to 3-h) discharge of ascospores, regardless of whether pseudothecia were incubated in constant light or dark. In the field, daily ascospore release increased between 1900 and 2300 h and again between 0700 and 1000 h local time. The release of ascospores occurred primarily during the early morning hours when relative humidity was decreasing and the canopy began to dry, or during evening hours when relative humidity was low and dew began to form. Few ascospores were released between 1100 and 1800 h when the bentgrass canopy was dry. The release of ascospores also was triggered by precipitation. Of the ascospores collected during precipitation events, 87% occurred within 10 h of the beginning of each event.

A PCR-Based Method for the Detection of Ophiosphaerella agrostis in Creeping Bentgrass.

Dead spot is a relatively new disease of creeping bentgrass and hybrid bermudagrass that is incited by Ophiosphaerella agrostis. Initial symptoms are difficult to diagnose and clinicians generally rely on the presence of pseudothecia within infected tissue or isolation of O. agrostis on an artificial medium. The main goal of this study was to develop a polymerase chain reaction-based technique capable of quickly identifying O. agrostis within infected creeping bentgrass tissues. Oligonucleotide primers specific for O. agrostis were developed based on the internal transcribed spacer (ITS) rDNA regions (ITS1 and ITS2) of three previously sequenced isolates of O. agrostis. The 22-bp primers amplified a 445- or 446-bp region of 80 O. agrostis isolates collected from creeping bentgrass and bermudagrass in 11 states. Primers did not amplify DNA from other common turfgrass pathogens, including three closely related species of Ophiosphaerella. Selective amplification of O. agrostis was successful from field-infected creeping bent-grass samples and primers did not amplify the DNA of noninfected, field-grown creeping bent-grass or hybrid bermudagrass plants. Amplification of purified O. agrostis DNA was successful at quantities between 50 ng and 5 pg. The entire process, including DNA isolation, amplification, and amplicon visualization, may be completed within 4 h. These results indicate the specificity of these primers for assisting in the accurate and timely identification of O. agrostis and the diagnosis of dead spot in both bentgrass and bermudagrass hosts.

Reactions in the Annual Medicago spp. Core Germ Plasm Collection to Phoma medicaginis.

The annual Medicago spp. core collection, consisting of 201 accessions, represents the genetic diversity inherent in 3,159 accessions from 36 annual Medicago spp. This germ plasm was evaluated for resistance to spring black stem and leaf spot caused by Phoma medicaginis. Spring black stem and leaf spot is a major destructive disease in perennial alfalfa (Medicago sativa) grown in North America, Europe, and other temperate regions. Disease control is based principally on the use of cultivars with moderate levels of resistance. Evaluation of the core collection was conducted using standardized environmental conditions in growth chambers, and included the M. sativa standard reference cultivars Ramsey (resistant) and Ranger (susceptible). The degree of resistance found among accessions within species was variable, but most annual species and accessions were susceptible. Most accessions from 10 species exhibited high disease resistance. These included accessions of M. constricta, M. doliata, M. heyniana, M. laciniata, M. lesinsii, M. murex, M. orbicularis, M. praecox, M. soleirolii, and M. tenoreana. Most of the accessions within M. arabica, M. minima, M. lanigera, M. rotata, M. rugosa, M. sauvagei, and M. scutellata were highly susceptible. Disease reactions among some accessions within species were highly variable. On a 0-to-5 disease severity scale, ratings ranged from 0.67 (PI 566873) to 4.29 (PI 566883) within accessions of M. polymorpha. Most of the M. truncatula accessions were susceptible, with a mean of 3.74. Resistant reactions were similar to those found in incompatible interactions with P. medicaginis and alfalfa, which have been associated with specific genes leading to the production of isoflavonoid phytoalexins. The large genetic variability in annual Medicago spp. offers potential for locating and utilizing disease resistance genes through breeding or genetic engineering that will enhance the utilization of Medicago spp. as a forage crop.

Molecular phylogeny of Leptosphaeria and Phaeosphaeria.

The objectives of this study were to determine the phylogenetic relationships of species of Leptosphaeria and Phaeosphaeria and evaluate the phylogenetic significance of morphological characters of the teleomorph, anamorph, and host. Sequences of the entire ITS region, including the 5.8S rDNA, of 59 isolates representing 54 species were analyzed and the phylogeny inferred using parsimony and distance analyses. Isolates grouped into three well-supported clades. The results of this study support the separation of Phaeosphaeria from Leptosphaeria sensu stricto. Leptosphaeria bicolor and the morphologically similar Leptosphaeria taiwanensis formed a separate, well-supported clade. We conclude that peridial wall morphology, anamorph characteristics, and to a lesser extent host, are phylogenetically significant at the generic level. Ascospore and conidial morphology are taxonomically useful at the species level.

Phylogeny of Stemphylium spp. based on ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences.

The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.

AFLP Comparisons Among Claviceps africana Isolates from the United States, Mexico, Africa, Australia, India, and Japan.

Eighty-seven isolates of the sorghum ergot pathogen, Claviceps africana, from diverse geographic locations were analyzed using four different amplified fragment length polymorphism (AFLP) primer combinations to determine genetic relationships among isolates. Most isolates showed unique AFLP haplotypes, indicating that substantial genetic variation is present within C. africana populations. Two major groupings of isolates were observable, with ca. 70% similarity between the two groups. One group consisted of Australian, Indian, and Japanese isolates and the other of U.S., Mexican, and African isolates. In spite of overall high levels of genetic diversity observed in C. africana, isolates within the two major groups were between 75 and 100% similar. The observed associations of C. africana isolates from worldwide sources could be the result of intercontinental trade and/or movement of seed. The data indicate that Africa was the likely source of C. africana that has become established in the Americas since 1996. Analysis of additional isolates in future studies will reveal whether these groupings are being maintained or whether population subdivision or reshuffling may occur.

Sources of Resistance to Anthracnose in the Annual Medicago Core Collection.

The annual genus Medicago core collection, consisting of 201 accessions, represents the genetic diversity inherent in 3,159 accessions from 36 annual Medicago species. This germ plasm was evaluated for resistance to anthracnose caused by Colletotrichum trifolii. Anthracnose is a major disease in perennial alfalfa (Medicago sativa L.) grown in North America and disease control is based principally on the use of resistant varieties. Evaluation of the core collection was conducted using standardized environmental conditions in growth chambers, and included the M. sativa standard reference cvs. Arc (resistant) and Saranac (susceptible). The degree of resistance found among accessions within species was highly variable; however, most annual species and accessions were susceptible. Only 14 accessions from seven species exhibited resistance greater than 40% seedling survival. These included accessions of M. murex, M. muricoleptis, M. polymorpha var. brevispina, M. polymorpha var. polymorpha, M. radiata, M. soleirolii, M. truncatula, and M. turbinata. Of the 12 accessions of M. polymorpha var. polymorpha, 4 exhibited more than 50% resistance, but 3 accessions were 100% susceptible. Most of the M. truncatula and M. turbinata accessions exhibited significantly more resistance than accessions of other species. Plant introduction (PI) accession number PI 495401 of M. muricoleptis exhibited 90.3% resistance. Accessions of M. scutellata were uniformly susceptible. Histological examinations of 14 of the most anthracnose-resistant accessions revealed that C. trifolii spores germinated and produced typical appressoria, but failed to penetrate and produce the primary and secondary hyphae characteristic of susceptible interactions. Resistant reactions were similar to those found in incompatible interactions with C. trifolii and alfalfa, which have been associated with specific genes leading to the production of isoflavonoid phytoalexins. The large genetic variability in annual Medicago spp. offers potential for locating and utilizing disease resistance genes through breeding or genetic engineering that will enhance the utilization of Medicago spp. as a forage crop.