Péter G. Ott

Bio-based resistance inducers for sustainable plant protection against pathogens

An increasing demand for environmentally acceptable alternative for traditional pesticides provides an impetus to conceive new bio-based strategies in crop protection. Employing induced resistance is one such strategy, consisting of boosting the natural plant immunity. Upon infections, plants defend themselves by activating their immune mechanisms. These are initiated after the recognition of an invading pathogen via the microbe-associated molecular patterns (MAMPs) or other microbe-derived molecules. Triggered responses inhibit pathogen spread from the infected site. Systemic signal transport even enables to prepare, i.e. prime, distal uninfected tissues for more rapid and enhanced response upon the consequent pathogen attack. Similar defense mechanisms can be triggered by purified MAMPs, pathogen-derived molecules, signal molecules involved in plant resistance to pathogens, such as salicylic and jasmonic acid, or a wide range of other chemical compounds. Induced resistance can be also conferred by plant-associated microorganisms, including beneficial bacteria or fungi. Treatment with resistance inducers or beneficial microorganisms provides long-lasting resistance for plants to a wide range of pathogens. This study surveys current knowledge on resistance and its mechanisms provided by microbe-, algae- and plant-derived elicitors in different crops. The main scope deals with bacterial substances and fungus-derived molecules chitin and chitosan and algae elicitors, including naturally sulphated polysaccharides such as ulvans, fucans or carageenans. Recent advances in the utilization of this strategy in practical crop protection are also discussed.

Novel Extracellular Chitinases Rapidly and Specifically Induced by General Bacterial Elicitors and Suppressed by Virulent Bacteria as a Marker of Early Basal Resistance in Tobacco

Early basal resistance (EBR, formerly known as early induced resistance) is triggered by general bacterial elicitors. EBR has been suggested to inhibit or retard expression of the type III secretion system of pathogenic bacteria and may also prevent nonpathogenic bacteria from colonizing the plant tissue. The quickness of EBR here plays a crucial role, compensating for a low bactericidal efficacy. This inhibitory activity should take place in the cell wall, as bacteria do not enter living plant cells. We found several soluble proteins in the intercellular fluid of tobacco leaf parenchyma that coincided with EBR under different environmental (light and temperature) conditions known to affect EBR. The two most prominent proteins proved to be novel chitinases (EC 3.2.1.14) that were transcriptionally induced before and during EBR development. Their expression in the apoplast was fast and not stress-regulated as opposed to many pathogenesis-related proteins. Nonpathogenic, saprophytic, and avirulent bacteria all induced EBR and the chitinases. Studies using these chitinases as EBR markers revealed that the virulent Pseudomonas syringae pv. tabaci, being sensitive to EBR, must suppress it while suppressing the chitinases. EBR, the chitinases, as well as their suppression are quantitatively related, implying a delicate balance determining the outcome of an infection.

Separation and detection of aflatoxins using overpressured-layer chromatography and bioautography

It has been established, by use of bioautographic detection with the phytopathogenic bacteria Pseudomonas savastanoi pv. phaseolicola, that the common aflatoxins have antimicrobial activity after OPLC separation. Our preliminary results suggest this antimicrobial activity originates from the activity of formaldehyde formed by the bacterial cells and/or from the methoxy groups of aflatoxin molecules.

Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry

Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid-liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products.

Separation and identification of antibacterial chamomile components using OPLC, bioautography and GC-MS

Components of 50% aqueous ethanol chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line overpressured layer chromatography (OPLC). This system consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected fractions were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% aqueous ethanol extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone.

Usefulness of Transgenic Luminescent Bacteria in Direct Bioautographic Investigation of Chamomile Extracts

Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile (Matricaria recutita) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

The influence of L-ascorbic acid on the antibacterial-toxic activity of aflatoxins on adsorbent layer.

Aims:  To substantiate the role of formaldehyde (HCHO) and its reaction products in the mechanism of the antibacterial‐toxic effect of aflatoxins B1 (AFB1), B2, G1 and G2.

Applicability of the BioArena System to Investigation of the Mechanisms of Biological Effects

Bioautography can be extended to a complex system called BioArena by linking different steps to it, for example dissolving a variety of compounds in the cell suspension to affect biological activity, measuring putative mediators of antibiosis, for example formaldehyde (HCHO) and hydrogen peroxide (H2O2) in the inoculated layer, and performing densitometric and ex and in situ spectroscopic examination to follow the changes in the inhibition zones and active compounds (e.g. antibiotics and toxins). Possibilities of the basic elements of BioArena system are illustrated in this paper by results with aflatoxin B1 (AFB1). Target bacterial cells in the logarithmic growth phase were found to be the most sensitive for direct bioautography. Densitometric signals of bioautograms (negative densitometry) of 0.125–1 μg AFB1 spots showed logarithmic correlation with the amount of AFB1. The HCHO capturer L-arginine decreased whereas the HCHO generator-mobilizer Cu(II) ions increased the antibacterial-toxic effect of AFB1. The latter effect was also confirmed by negative densitometry. Besides higher levels of HCHO, a decrease of H2O2 in the toxin spot was found. HCHO could also originate, among other sources, from demethylation of AFB1, which is apparent from the Fourier transform Raman spectra obtained in situ from the AFB1-containing spots. These results support the suggested role of HCHO and its reaction products with H2O2 (e.g. singlet oxygen (1O2), ozone (O3)) in the antibacterial-toxic effect of AFB1.

BioArena: An unlimited possibility of biochemical interactions in the adsorbent layer after chromatographic separation

The BioArena system, which integrates the modern technique and biological results of bioautography with layer liquid chromatography is especially suitable for investigating biochemical interactions. Formaldehyde (HCHO) and its reaction products play a crucial role in the antibiotic activity of trans-resveratrol and other molecules–when HCHO-capturing molecules are used in culture media the antimicrobial activity of antibiotic-like compounds decreases substantially. HCHO and hydrogen peroxide are present as normal endogenous compounds in cells, so there is a possibility of interaction in which singlet oxygen (1O2) and excited HCHO can be formed. The 1O2 can oxidize water molecules and so H2O3 can be formed, from which, by disproportion, among other reactions, ozone (O3) also can be formed in the chromatographic spots. Elimination of HCHO and/or O3 from the spots results in a decrease in the antiproliferative effect.

The potential of BioArena in the study of the formaldehydome

New results with BioArena as a complex separation and detection (evaluation) system support earlier observations that formaldehyde (HCHO) and its reaction products play a special and crucial role in the effects of antibiotic in general. It has been established that antibiotic-like compounds (e.g. trans-resveratrol, Cu(II) ions) have a duplicate inhibiting effect on pathogen cells as a result of the action of HCHO. HCHO as a key molecule of the formaldehydome participates in series of interactions which can be screened by means of different spectroscopic and chromatographic techniques; accumulation of HCHO and its reaction products in TLC spots is limited, however, so indirect detection is advantageous. In BioArena the planar stationary phase bed after TLC/HPTLC and, mainly, OPLC separation can be used for manifestation of deprivation of HCHO from antibiotic spots, for observation of the duplicate effect of substances with a direct effect, and for demonstration of cell proliferation promotion and retardation.