Two Non-Necrotic Disease Resistance Types Distinctly Affect the Expression of Key Pathogenic Determinants of Xanthomonas euvesicatoria in Pepper.

Pepper (Capsicum annuum L.) carrying the gds (corresponding to bs5) gene can prevent the development of bacterial leaf spot disease without HR. However, little is known regarding the development of the resistance mechanism encoded by gds, especially its influence on the bacterium. Here, the effect of gds was compared with pattern-triggered immunity (PTI), another form of asymptomatic resistance, to reveal the interactions and differences between these two defense mechanisms. The level of resistance was examined by its effect on the bacterial growth and in planta expression of the stress and pathogenicity genes of Xanthomonas euvesicatoria. PTI, which was activated with a Pseudomonas syringae hrcC mutant pretreatment, inhibited the growth of Xanthomonas euvesicatoria to a greater extent than gds, and the effect was additive when PTI was activated in gds plants. The stronger influence of PTI was further supported by the expression pattern of the dpsA bacterial stress gene, which reached its highest expression level in PTI-induced plants. PTI inhibited the hrp/hrc expression, but unexpectedly, in gds plant leaves, the hrp/hrc genes were generally expressed at a higher level than in the susceptible one. These results imply that different mechanisms underlie the gds and PTI to perform the symptomless defense reaction.

Goldenrod Root Compounds Active against Crop Pathogenic Fungi.

Root extracts of three goldenrods were screened for antimicrobial compounds. 2Z,8Z- and 2E,8Z-matricaria esters from European goldenrod (Solidago virgaurea) and E- and Z-dehydromatricaria esters from grass-leaved goldenrod (Solidago graminifolia) and first from showy goldenrod (Solidago speciosa) were identified by high-performance thin-layer chromatography combined with effect-directed analysis and high-resolution mass spectrometry or nuclear magnetic resonance spectroscopy after liquid chromatographic fractionation and isolation. Next to their antibacterial effects (against Bacillus subtilis, Aliivibrio fischeri, and Pseudomonas syringae pv. maculicola), they inhibited the crop pathogenic fungi Fusarium avenaceum and Bipolaris sorokiniana with half maximal inhibitory concentrations (IC50) between 31 and 107 µg/mL. Benzyl 2-hydroxy-6-methoxybenzoate, for the first time found in showy goldenrod root, showed the strongest antifungal effect, with IC50 of 25-26 µg/mL for both fungal strains.

Acetylcholinesterase inhibitors in the giant goldenrod root.

Eight bioactive clerodane diterpenes from the root extract of Solidago gigantea Ait. (giant goldenrod) were quantified by high-performance thin-layer chromatography (HPTLC) and two newly developed hyphenated methods. One uses vanillin sulphuric acid derivatization and densitometry, and the other an inhibition assay of acetylcholinesterase (AChE) and video densitometry. Both methods gave figures of merit for quantification including 5.8-33.9 ng and 175.5-448.7 ng LOQs and 2.7-6.9 RSD% and 8.8-13.9 RSD% inter-day precisions, respectively. Based on the diterpenes' content of 14 root samples collected over a year from the same plant population, the fully flowering plant is suggested to collect the root as a source of these compounds. Excepting one diterpene (with the lowest retardation factor), the quantitative results for the richest sample obtained by the two methods were in harmony. The difference could be due to a matrix effect.

Exposure to Batrachochytrium dendrobatidis affects chemical defences in two anuran amphibians, Rana dalmatina and Bufo bufo.

BACKGROUND: Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). RESULTS: Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. CONCLUSIONS: The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events.

Bioactive clerodane diterpenes of giant goldenrod (Solidago gigantea Ait.) root extract.

Giant goldenrod (Solidago gigantea Ait.) root extract was screened for bioactive compounds by high-performance thin-layer chromatography (HPTLC), coupled with effect-directed analysis including antibacterial (Bacillus subtilis F1276, B. subtilis subsp. spizizenii, Aliivibrio fischeri and Xanthomonas euvesicatoria), antifungal (Fusarium avenaceum) and enzyme inhibition (acetyl- and butyrylcholinesterases, α- and ß-glucosidases and α-amylase) assays. Compounds of six multipotent zones (Sg1-Sg6) were characterized by HPTLC-heated electrospray ionization-high-resolution mass spectrometry (HRMS) and HPTLC-Direct Analysis in Real Time-HRMS. Apart from zone Sg3, containing three compounds, a single characteristic compound was detectable in each bioactive zone. The bioassay-guided isolation using preparative-scale flash chromatography and high-performance liquid chromatography provided eight compounds that were identified by NMR spectroscopy as clerodane diterpenes. All isolates possessed inhibiting activity against at least one of the tested microorganisms.

Relationships Between Chemical Defenses of Common Toad (Bufo bufo) Tadpoles and Bacterial Community Structure of their Natural Aquatic Habitat.

Many organisms synthesize secondary metabolites against natural enemies. However, to which environmental factors the production of these metabolites is adjusted to is poorly investigated in animals, especially so in vertebrates. Bufadienolides are steroidal compounds that are present in a wide range of plants and animals and, if present in large quantities, can provide protection against natural enemies, such as pathogens. In a correlative study involving 16 natural populations we investigated how variation in bufadienolide content of larval common toads (Bufo bufo) is associated with the bacterial community structure of their aquatic environment. We also evaluated pond size, macrovegetation cover, and the abundance of predators, conspecifics and other larval amphibians. We measured toxin content of tadpoles using HPLC-MS and determined the number of bufadienolide compounds (NBC) and the total quantity of bufadienolides (TBQ). AICc-based model selection revealed strong relationships of NBC and TBQ with bacterial community structure of the aquatic habitat as well as with the presence of conspecific tadpoles. The observed relationships may have arisen due to adaptation to local bacterial communities, phenotypic plasticity, differential biotransformation of toxin compounds by different bacterial communities, or a combination of these processes. Bacterial groups that contribute to among-population variation in toxin content remain to be pinpointed, but our study suggesting that toxin production may be influenced by the bacterial community of the environment represents an important step towards understanding the ecological and evolutionary processes leading to microbiota-mediated variation in skin toxin profiles of aquatic vertebrates.

Distinction and valorization of 30 root extracts of five goldenrod (Solidago) species.

A high-performance thin-layer chromatography (HPTLC) method was developed for rapid and easy-to-perform discrimination between five goldenrod species present in Europe: the native Solidago virgaurea and the four invasive aliens, S. canadensis, S. gigantea, S. rugosa and S. graminifolia. The chemotaxonomic distinction was based on the chemical profile of their root extracts, confirmed by principal component analysis. This allowed the distinction of the goldenrods in wintertime, when classical morphological methods are not applicable. Their enzyme inhibitory profiles were determined by HPTLC combined with α-glucosidase, ß-glucosidase, α-amylase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) assays. Two compounds of S. canadensis showed the most intense enzyme inhibition in all assays, having also antibacterial activity against Bacillus subtilis, Xanthomonas euvesicatoria and Aliivibrio fischeri strains. HPTLC-high-resolution mass spectrometry (HRMS), bioassay-guided isolation, NMR spectroscopy and literature data led to the characterization and identification of the labdane diterpenes solidagenone and presolidagenone as the active S. canadensis root components. The previously identified polyacetylenes (2Z,8Z and 2E,8Z matricaria esters) of S. virgaurea, also inhibited all enzymes. Except for the known anti-AChE effect of the 2Z,8Z-matricaria ester, this is the first report on the α-glucosidase, ß-glucosidase, α-amylase, AChE and BChE inhibitory activity of these potent compounds. The anti-hyperglycemic effects of the S. canadensis labdanoids were also observed for the first time. Combined with effect-directed assays and HRMS, hyphenated HPTLC allowed an effect-directed high-throughput screening and a fast characterization of multipotent compounds. The investigation of botanicals by fast, hyphenated, bioanalytical tools substantially increased the information gain with regard to active principles (bioprofiling) and efficiently pointed to potent candidates for drug development.

Chemical defense of toad tadpoles under risk by four predator species.

Many organisms use inducible defenses as protection against predators. In animals, inducible defenses may manifest as changes in behavior, morphology, physiology, or life history, and prey species can adjust their defensive responses based on the dangerousness of predators. Analogously, prey may also change the composition and quantity of defensive chemicals when they coexist with different predators, but such predator-induced plasticity in chemical defenses remains elusive in vertebrates. In this study, we investigated whether tadpoles of the common toad (Bufo bufo) adjust their chemical defenses to predation risk in general and specifically to the presence of different predator species; furthermore, we assessed the adaptive value of the induced defense. We reared tadpoles in the presence or absence of one of four caged predator species in a mesocosm experiment, analyzed the composition and quantity of their bufadienolide toxins, and exposed them to free-ranging predators. We found that toad tadpoles did not respond to predation risk by upregulating their bufadienolide synthesis. Fishes and newts consumed only a small percentage of toad tadpoles, suggesting that bufadienolides provided protection against vertebrate predators, irrespective of the rearing environment. Backswimmers consumed toad tadpoles regardless of treatment. Dragonfly larvae were the most voracious predators and consumed more predator-naïve toad tadpoles than tadpoles raised in the presence of dragonfly cues. These results suggest that tadpoles in our experiment had high enough toxin levels for an effective defense against vertebrate predators even in the absence of predator cues. The lack of predator-induced phenotypic plasticity in bufadienolide synthesis may be due to local adaptation for constantly high chemical defense against fishes in the study population and/or due to the high density of conspecifics.

Isolation and structural elucidation of a novel brunnein-type antioxidant ß-carboline alkaloid from Cyclocybe cylindracea.

Effect-directed isolation of free radical scavengers from the methanol extract of the freeze-dried fruiting bodies of the cultivated basidiomycetous mushroom, black poplar (Cyclocybe cylindracea), yielded a ß-carboline alkaloid. Its structure was determined based on ESI-TOF-MS/MS, NMR and circular dichroism spectra by comparison with published data. The compound, identified as the C1-S diastereomer of brunnein B, exhibited explicit radical scavenging activity (EC50 = 119.1 ±â€¯1.2 µg/mL). The quantity of the active component was determined with HPLC-MS in the fruiting body (36.2 ±â€¯2.8 ng/g DW, dry weight) and its different tissues such as peel (94.7 ±â€¯1.9 ng/g DW), inner cap (90.5 ±â€¯1.3 ng/g DW), gills (71.5 ±â€¯0.6 ng/g DW), and stipe (162.2 ±â€¯1.7 ng/g DW). It is a ß-carboline alkaloid that was not reported previously.

Layer chromatography-bioassays directed screening and identification of antibacterial compounds from Scotch thistle.

The antibacterial profiling of Onopordum acanthium L. leaf extract and subsequent targeted identification of active compounds is demonstrated. Thin-layer chromatography (TLC) and off-line overpressured layer chromatography (OPLC) coupled with direct bioautography were utilized for investigation of the extract against eight bacterial strains including two plant and three human pathogens and a soil, a marine and a probiotic human gut bacteria. Antibacterial fractions obtaining infusion-transfusion OPLC were transferred to HPLC-MS/MS analysis that resulted in the characterization of three active compounds and two of them were identified as, linoleic and linolenic acid. OPLC method was adopted to preparative-scale flash chromatography for the isolation of the third active compound, which was identified after a further semi-preparative HPLC purification as the germacranolide sesquiterpene lactone onopordopicrin. Pure onopordopicrin exhibited antibacterial activity that was specified as minimal inhibitory concentration in the liquid phase as well.

Chronic exposure to a glyphosate-based herbicide makes toad larvae more toxic.

Chemical pollutants can exert various sublethal effects on wildlife, leading to complex fitness consequences. Many animals use defensive chemicals as protection from predators and diseases, yet the effects of chemical contaminants on this important fitness component are poorly known. Understanding such effects is especially relevant for amphibians, the globally most threatened group of vertebrates, because they are particularly vulnerable to chemical pollution. We conducted two experiments to investigate how exposure to glyphosate-based herbicides, the most widespread agrochemicals worldwide, affects the production of bufadienolides, the main compounds of chemical defence in common toads (Bufo bufo). In both experiments, herbicide exposure increased the amount of bufadienolides in toad tadpoles. In the laboratory, individuals exposed to 4 mg a.e./L glyphosate throughout their larval development had higher bufadienolide content at metamorphosis than non-exposed tadpoles, whereas exposure for 9 days to the same concentration or to 2 mg a.e./L throughout larval development or for 9 days had no detectable effect. In outdoor mesocosms, tadpoles from 16 populations exhibited elevated bufadienolide content after three-weeks exposure to both concentrations of the herbicide. These results show that pesticide exposure can have unexpected effects on non-target organisms, with potential consequences for the conservation management of toxin-producing species and their predators.

Age- and environment-dependent changes in chemical defences of larval and post-metamorphic toads.

BACKGROUND: Chemical defences are widespread in animals, but how their production is adjusted to ecological conditions is poorly known. Optimal defence theory predicts that inducible defences are favoured over constitutive defences when toxin production is costly and the need for it varies across environments. However, if some environmental changes occur predictably (e.g. coupled to transitions during ontogeny), whereas others are unpredictable (e.g. predation, food availability), changes in defences may have constitutive as well as plastic elements. To investigate this phenomenon, we raised common toad (Bufo bufo) tadpoles with ad libitum or limited food and in the presence or absence of chemical cues on predation risk, and measured their toxin content on 5 occasions during early ontogeny. RESULTS: The number of compounds showed limited variation with age in tadpoles and was unaffected by food limitation and predator cues. The total amount of bufadienolides first increased and later decreased during development, and it was elevated in young and mid-aged tadpoles with limited food availability compared to their ad libitum fed conspecifics, whereas it did not change in response to cues on predation risk. We provide the first evidence for the active synthesis of defensive toxin compounds this early during ontogeny in amphibians. Furthermore, the observation of increased quantities of bufadienolides in food-restricted tadpoles is the first experimental demonstration of resource-dependent induction of elevated de novo toxin production, suggesting a role for bufadienolides in allelopathy. CONCLUSIONS: Our study shows that the evolution of phenotypic plasticity in chemical defences may depend on the ecological context (i.e. predation vs. competition). Our results furthermore suggest that the age-dependent changes in the diversity of toxin compounds in developing toads may be fixed (i.e., constitutive), timed for the developmental stages in which they are most reliant on their chemical arsenal, whereas inducible plasticity may prevail in the amount of synthesized compounds.

Effect-Directed Discovery of Bioactive Compounds Followed by Highly Targeted Characterization, Isolation and Identification, Exemplarily Shown for Solidago virgaurea.

A nontargeted, effect-directed screening (bioprofiling) and a subsequent highly targeted characterization of antibacterial compounds from plant matrices is demonstrated on the example of Solidago virgaurea root extracts. The procedure comprises high-performance thin-layer chromatography (HPTLC) coupled with six bacterial bioassays including two plant pathogens, a radical scavenging assay, an acetylcholinesterase assay as well as in situ and ex situ mass spectrometric analyses. In situ mass spectra were directly recorded from the adsorbent using the Direct Analysis in Real Time interface (HPTLC-DART-MS), whereas ex situ mass spectra were recorded using an elution head-based interface (HPTLC-ESI-MS). For further bioassay-guided isolation of the main antimicrobial compounds, flash chromatographic fractionation and semipreparative high-performance liquid chromatographic purification were used and nuclear magnetic resonance data allowed the identification of the unknown antimicrobial compounds as 2Z,8Z- and 2E,8Z-matricaria esters. The discovered antibacterial activity was confirmed and specified by a luminometric assay and as minimal inhibitory concentration in the liquid phase.

Variation in Chemical Defense Among Natural Populations of Common Toad, Bufo bufo, Tadpoles: the Role of Environmental Factors.

Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles' toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.

Rapid, Bioassay-Guided Process for the Detection and Identification of Antibacterial Neem Oil Compounds.

Bioassay guidance was used along the whole process including method development, isolation and identification of antibacterial neem (Azadirachta indica) oil compounds. The biomonitoring was performed by direct bioautography (DB), a combination of thin-layer chromatography (TLC) and antimicrobial detection. DB of neem oil showed one antibacterial zone that was not UV-active; therefore, the TLC separation was improved under DB control. The chromatographic zone that exhibited activity against Bacillus subtilis, Xanthomonas euvesicatoria, Aliivibrio fischeri, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus was characterized by TLC reagents, indicating a lipophilic, fatty acid-like chemical feature. Two compounds were found and identified in the active zone by high-performance liquid chromatography-electrospray ionization mass spectrometry as linoleic and oleic acids. Both fatty acids inhibited B. subtilis, but A. fischeri was sensitive only against linoleic acid.

Nitrate content in a collection of higher mushrooms.

BACKGROUND: Data of mushroom nitrate content from scientific studies is limited. There have been two such recent investigations (mainly regarding certain cultivated species). To obtain comparable analytical data, we analyzed 134 samples of 54 taxa gathered and prepared by our department. RESULTS: The mushroom species were evaluated according to their nutritional types: saprotrophic, mycorrhizal and wood-decaying groups. Low and relatively invariable contents were found in the mycorrhizal (216.5 mg kg(-1) dry matter (DM) and wood-decaying groups (228.6 mg kg(-1) DM), but in the saprotrophic group we observed a wide variability (151.4-12 715 mg kg(-1) DM). CONCLUSION: A considerable nitrate content was found in samples of seven 'accumulator' species (Clitocybe nebularis, C. odora, Lepista nuda, L. personata, L. irina, Macrolepiota rachodes and M. procera). The toxicological relevance of daily uptake of acceptable nitrate content via mushrooms only is not presumable, but the 'accumulator' saprotrophic species can be 'contributors' to our nitrate intake in foods.