Heat Treatments at Varying Ambient Temperatures and Durations Differentially Affect Plant Defense to Blumeria hordei in a Resistant and a Susceptible Hordeum vulgare Line.

Our previous research showed that a powdery mildew resistant barley line (MvHV07-17) maintains its resistance to Blumeria hordei (Bh) even if plants are exposed to a long-term high temperature of 35°C for 120 h before Bh inoculation, whereas such high temperature pretreatment further increases susceptibility to infection in the susceptible barley line MvHV118-17. In the present study, we extended this approach using short-term high-temperature water treatment (49°C for 30 s) to determine how it affects powdery mildew resistance in these barley lines. We found that this short-term heat shock (HS) impaired plant defense responses, as reflected by development of Bh colonies and visible necrotic spots on leaves of MvHV07-17, which does not develop visible symptoms upon Bh inoculation under optimal growth conditions. In contrast, both HS and long-term heat stress enhanced susceptibility to Bh in MvHV118-17 plants. These results were supported by the measurement of Bh biomass using a qPCR method. Furthermore, microscopic examinations showed that HS elevated the rate of successful Bh penetration events and the spread of cell death in the surrounding mesophyll area and allowed for colony formation and sporulation in resistant barley, whereas early and effective plant defense responses, such as papilla formation and single-cell epidermal hypersensitive response, were significantly reduced. Furthermore, we found that the accumulation of hydrogen peroxide in both resistant and susceptible barley was correlated with susceptibility induced by HS and long-term heat-stress. This study may contribute to a better understanding of plant defense responses to Bh in barley exposed to heat. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Enhanced Resistance to Viruses in Nicotiana edwardsonii 'Columbia' Is Dependent on Salicylic Acid, Correlates with High Glutathione Levels, and Extends to Plant-Pathogenic Bacteria and Abiotic Stress.

Our earlier research showed that an interspecific tobacco hybrid (Nicotiana edwardsonii 'Columbia' [NEC]) displays elevated levels of salicylic acid (SA) and enhanced resistance to localized necrotic symptoms (hypersensitive response [HR]) caused by tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV), as compared with another interspecific hybrid (Nicotiana edwardsonii [NE]) derived from the same parents. In the present study, we investigated whether symptomatic resistance in NEC is indeed associated with the inhibition of TMV and TNV and whether SA plays a role in this process. We demonstrated that enhanced viral resistance in NEC is manifested as both milder local necrotic (HR) symptoms and reduced levels of TMV and TNV. The presence of an adequate amount of SA contributes to the enhanced defense response of NEC to TMV and TNV, as the absence of SA resulted in seriously impaired viral resistance. Elevated levels of subcellular tripeptide glutathione (GSH) in NEC plants in response to viral infection suggest that in addition to SA, GSH may also contribute to the elevated viral resistance of NEC. Furthermore, we found that NEC displays an enhanced resistance not only to viral pathogens but also to bacterial infections and abiotic oxidative stress induced by paraquat treatments. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Antibacterial effect of essential oils and their components against Xanthomonas arboricola pv. pruni revealed by microdilution and direct bioautographic assays.

Bacterial spot of stone fruits caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most significant diseases of several Prunus species. Disease outbreaks can result in severe economic losses while the control options are limited. Antibacterial efficacy of essential oils (EOs) of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm was assessed against two Hungarian Xap isolates. The minimal inhibitory concentration (MIC) was determined by broth microdilution assay and for the identification of active EOs' components a newly introduced high-performance thin-layer chromatography (HPTLC)-Xap (direct bioautography) method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) was applied. All EOs inhibited both bacterium isolates, but cinnamon proved to be the most effective EO with MIC values of 31.25 µg/mL and 62.5 µg/mL, respectively. Compounds in the antibacterial HPTLC zones were identified as thymol in thyme, trans-cinnamaldehyde in cinnamon, eugenol in clove, borneol in rosemary, terpinen-4-ol in tea tree, citral (neral and geranial) in lemon grass and lemon balm, and citronellal and nerol in citronella grass. Regarding active compounds, thymol had the highest efficiency with a MIC value of 50 µg/mL. Antibacterial effects of EOs have already been proven for several Xanthomonas species, but to our knowledge, the studied EOs, except for lemon grass and eucalyptus, were tested for the first time against Xap. Furthermore, in case of Xap, this is the first report demonstrating that direct bioautography is a fast and suitable method for screening anti-Xap components of complex matrices, like EOs.

Antimicrobial Diterpenes from Rough Goldenrod (Solidago rugosa Mill.).

Solidago rugosa is one of the goldenrod species native to North America but has sporadically naturalized as an alien plant in Europe. The investigation of the root and leaf ethanol extracts of the plant using a bioassay-guided process with an anti-Bacillus assay resulted in the isolation of two antimicrobial components. Structure elucidation was performed based on high-resolution tandem mass spectrometric and one- and two-dimensional NMR spectroscopic analyses that revealed (-)-hardwickiic acid (Compound 1) and (-)-abietic acid (Compound 2). The isolates were evaluated for their antimicrobial properties against several plant pathogenic bacterial and fungal strains. Both compounds demonstrated an antibacterial effect, especially against Gram-positive bacterial strains (Bacillus spizizenii, Clavibacter michiganensis subsp. michiganensis, and Curtobacterium flaccumfaciens pv. flaccumfaciens) with half maximal inhibitory concentration (IC50) between 1 and 5.1 µg/mL (5-20 times higher than that of the positive control gentamicin). In the used concentrations, minimal bactericidal concentration (MBC) was reached only against the non-pathogen B. spizizenii. Besides their activity against Fusarium avenaceum, the highest antifungal activity was observed for Compound 1 against Bipolaris sorokiniana with an IC50 of 3.8 µg/mL.

Near-Isogenic Barley Lines Show Enhanced Susceptibility to Powdery Mildew Infection Following High-Temperature Stress.

Barley cultivation is adversely affected by high-temperature stress, which may modulate plant defense responses to pathogens such as barley powdery mildew (Blumeria graminis f. sp. hordei, Bgh). Earlier research focused mainly on the influence of short-term heat stress (heat shock) of barley on Bgh infection. In this study, our aim was to investigate the effects of both short- and long-term heat stress (35 °C from 30 s to 5 days) on Bgh infection in the barley cultivar Ingrid and its near-isogenic lines containing different powdery mildew resistance genes (Mla12, Mlg, and mlo5) by analyzing symptom severity and Bgh biomass with RT-qPCR. The expression of selected barley defense genes (BAX inhibitor-1, Pathogenesis- related protein-1b, Respiratory burst oxidase homologue F2, and Heat shock protein 90-1) was also monitored in plants previously exposed to heat stress followed by inoculation with Bgh. We demonstrated that pre-exposure to short- and long-term heat stress negatively affects the resistance of all resistant lines manifested by the appearance of powdery mildew symptoms and increased Bgh biomass. Furthermore, prolonged heat stress (48 and 120 h) enhanced both Bgh symptoms and biomass in susceptible wild-type Ingrid. Heat stress suppressed and delayed early defense gene activation in resistant lines, which is a possible reason why resistant barley became partially susceptible to Bgh.

Heat Stress Pre-Exposure May Differentially Modulate Plant Defense to Powdery Mildew in a Resistant and Susceptible Barley Genotype.

Heat stress negatively affects barley production and under elevated temperatures defense responses to powdery mildew (Blumeria graminis f. sp. hordei, Bgh) are altered. Previous research has analyzed the effects of short-term (30 s to 2 h) heat stress, however, few data are available on the influence of long-term exposure to heat on powdery mildew infections. We simultaneously assessed the effects of short and long term heat pre-exposure on resistance/susceptibility of barley to Bgh, evaluating powdery mildew infection by analyzing symptoms and Bgh biomass with RT-qPCR in barley plants pre-exposed to high temperatures (28 and 35 °C from 30 s to 5 days). Plant defense gene expression after heat stress pre-exposure and inoculation was also monitored. Our results show that prolonged heat stress (24, 48 and 120 h) further enhanced Bgh susceptibility in a susceptible barley line (MvHV118-17), while a resistant line (MvHV07-17) retained its pathogen resistance. Furthermore, prolonged heat stress significantly repressed the expression of several defense-related genes (BAX inhibitor-1, Pathogenesis related-1b and Respiratory burst oxidase homologue F2) in both resistant and susceptible barley lines. Remarkably, heat-suppressed defense gene expression returned to normal levels only in MvHV07-17, a possible reason why this barley line retains Bgh resistance even at high temperatures.

A pattern-triggered immunity-related phenolic, acetosyringone, boosts rapid inhibition of a diverse set of plant pathogenic bacteria.

BACKGROUND: Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix). RESULTS: We detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others' previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC4(3). CONCLUSIONS: Level of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

Reactive Oxygen Species Contribute to Symptomless, Extreme Resistance to Potato virus X in Tobacco.

Here we show that in tobacco (Nicotiana tabacum cultivar Samsun NN Rx1) the development of Rx1 gene-mediated, symptomless, extreme resistance to Potato virus X (PVX) is preceded by an early, intensive accumulation of the reactive oxygen species (ROS) superoxide (O2·-), evident between 1 and 6 h after inoculation and associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities. This suggests a direct contribution of this ROS to virus restriction during symptomless, extreme resistance. Superoxide inhibition in PVX-inoculated leaves by infiltration of antioxidants (superoxide dismutase [SOD] and catalase [CAT]) partially suppresses extreme resistance in parallel with the appearance of localized leaf necrosis resembling a hypersensitive resistance (HR) response. F1 progeny from crosses of Rx1 and ferritin overproducer (deficient in production of the ROS OH·) tobaccos also display a suppressed extreme resistance to PVX, because significantly increased virus levels are coupled to HR, suggesting a role of the hydroxyl radical (OH·) in this symptomless antiviral defense. In addition, treatment of PVX-susceptible tobacco with a superoxide-generating agent (riboflavin/methionine) results in HR-like symptoms and reduced PVX titers. Finally, by comparing defense responses during PVX-elicited symptomless, extreme resistance and HR-type resistance elicited by Tobacco mosaic virus, we conclude that defense reactions typical of an HR (e.g., induction of cell death/ROS-regulator genes and antioxidants) are early and transient in the course of extreme resistance. Our results demonstrate the contribution of early accumulation of ROS (superoxide, OH·) in limiting PVX replication during symptomless extreme resistance and support earlier findings that virus-elicited HR represents a delayed, slower resistance response than symptomless, extreme resistance.

Superoxide (O2.-) accumulation contributes to symptomless (type I) nonhost resistance of plants to biotrophic pathogens.

Nonhost resistance is the most common form of disease resistance exhibited by plants against most pathogenic microorganisms. Type I nonhost resistance is symptomless (i.e. no macroscopically visible cell/tissue death), implying an early halt of pathogen growth. The timing/speed of defences is much more rapid during type I nonhost resistance than during type II nonhost and host ("gene-for-gene") resistance associated with a hypersensitive response (localized necrosis, HR). However, the mechanism(s) underlying symptomless (type I) nonhost resistance is not entirely understood. Here we assessed accumulation dynamics of the reactive oxygen species superoxide (O2.-) during interactions of plants with a range of biotrophic and hemibiotrophic pathogens resulting in susceptibility, symptomless nonhost resistance or host resistance with HR. Our results show that the timing of macroscopically detectable superoxide accumulation (1-4 days after inoculation, DAI) is always associated with the speed of the defense response (symptomless nonhost resistance vs. host resistance with HR) in inoculated leaves. The relatively early (1 DAI) superoxide accumulation during symptomless nonhost resistance of barley to wheat powdery mildew (Blumeria graminis f. sp. tritici) is localized to mesophyll chloroplasts of inoculated leaves and coupled to enhanced NADPH oxidase (EC 1.6.3.1) activity and transient increases in expression of genes regulating superoxide levels and cell death (superoxide dismutase, HvSOD1 and BAX inhibitor-1, HvBI-1). Importantly, the partial suppression of symptomless nonhost resistance of barley to wheat powdery mildew by heat shock (49 °C, 45 s) and antioxidant (SOD and catalase) treatments points to a functional role of superoxide in symptomless (type I) nonhost resistance.

Description of the Nicotiana benthamiana-Cercospora nicotianae Pathosystem.

Nicotiana benthamiana is a valuable model organism in plant biology research. This report describes its extended applicability in the field of molecular plant pathology by introducing a nonbiotrophic fungal pathogen Cercospora nicotianae that can be conveniently used under laboratory conditions, consistently induces a necrotic leaf spot disease on Nicotiana benthamiana, and is specialized on solanaceous plants. Our inoculation studies showed that C. nicotianae more effectively colonizes N. benthamiana than its conventional host, N. tabacum. The functions of two critical regulators of host immunity, coronatine-insensitive 1 (COI1) and ethylene-insensitive 2 (EIN2), were studied in N. benthamiana using Tobacco rattle virus-based virus-induced gene silencing (VIGS). Perturbation of jasmonic acid or ethylene signaling by VIGS of either COI1 or EIN2, respectively, resulted in markedly increased Cercospora leaf spot symptoms on N. benthamiana plants. These results suggest that the N. benthamiana-C. nicotianae host-pathogen interaction is a prospective but hitherto unutilized pathosystem for studying gene functions in diseased plants.

Regulatory Proteolysis in Arabidopsis-Pathogen Interactions.

Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available.

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants.

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.