Involvement of oxidative stress in localization of bacterial spot infection in pepper plants.

Xanthomonas euvesicatoria is a major cause of bacterial spot disease in various crops. The present study was focused on the pathosystem pepper (Capsicum annuum L.) - X. euvesicatoria 269p (wild strain). The infectious process was studied using several different modes of in vivo inoculation under controlled conditions. The spread of the pathogen in different parts of the plants was monitored by a new qPCR procedure developed for the detection of X. euvesicatoria, as well as by re-isolation of viable bacterial cells. Photosynthesis, the number of viable pathogens, oxidative stress markers, activities of the main antioxidant enzymes, and levels of nonenzymatic antioxidants in the novel single-leaf model system were studied. The most important observation is that the invasion of the pathogen causes local infection and the dissemination of bacteria to the healthy parts of the host is blocked. The plants limit bacterial colonization around the entry points. Oxidative burst and alterations in antioxidant defenses are detected in infectious leaf lesions. Localized ROS overproduction resembles a hypersensitive response, but several differences can be observed. We assumed that pepper plants are more likely to manifest an intermediate phenotype, similar to lesions simulating disease or leaf flecking. By localizing the infection, possibly involving oxidative stress, the plant survives. However, the same applies to bacteria. The pathogen multiplies at the infection spots and is transmitted to other plants. Our conclusion is that the intermediate phenotype in the studied pathosystem is an example of long and successful co-evolution for both species.

Xanthomonas euvesicatoria-Specific Bacteriophage BsXeu269p/3 Reduces the Spread of Bacterial Spot Disease in Pepper Plants.

The present study was focused on the pathosystem pepper plants (Capsicum annuum L.)-phytopathogenic bacterium X. euvesicatoria (wild strain 269p)-bacteriophage BsXeu269p/3 and the possibility of bacteriophage-mediated biocontrol of the disease. Two new model systems were designed for the monitoring of the effect of the phage treatment on the infectious process in vivo. The spread of the bacteriophage and the pathogen was monitored by qPCR. A new pair of primers for phage detection via qPCR was designed, as well as probes for TaqMan qPCR. The epiphytic bacterial population and the potential bacteriolytic effect of BsXeu269p/3 in vivo was observed by SEM. An aerosol-mediated transmission model system demonstrated that treatment with BsXeu269p/3 reduced the amount of X. euvesicatoria on the leaf surface five-fold. The needle-pricking model system showed a significant reduction of the amount of the pathogen in infectious lesions treated with BsXeu269p/3 (av. 59.7%), compared to the untreated control. We found that the phage titer is 10-fold higher in the infection lesions but it was still discoverable even in the absence of the specific host in the leaves. This is the first report of in vivo assessment of the biocontrol potential of locally isolated phages against BS pathogen X. euvesicatoria in Bulgaria.

Biochemical Alterations in Triticale Seedlings Pretreated with Selective Herbicide and Subjected to Drought or Waterlogging Stress.

Waterlogging and drought disrupt crop development and productivity. Triticale is known to be relatively tolerant to different stress factors. In natural conditions, plants are rather subjected to multiple environmental factors. Serrate® (Syngenta) is a systemic selective herbicide suitable for cereal crops such as triticale and wheat to restrain annual grass and broadleaf weeds. Triticale (×Triticosecale Wittm., cv. Rozhen) was grown as soil culture under controlled conditions. Seventeen-day-old plantlets were leaf sprayed with Serrate®. The water stress (drought or waterlogging) was applied after 72 h for 7 days, and then the seedlings were left for recovery. The herbicide does not provoke sharp alterations in the antioxidant state (stress markers level, and antioxidant and xenobiotic-detoxifying enzymes activity). The water stresses and combined treatments enhanced significantly the content of stress markers (malondialdehyde, proline, hydrogen peroxide), non-enzymatic (total phenolics and thiol groups-containing compounds), and enzymatic (activities of superoxide dismutase, catalase, guaiacol peroxidase, glutathione reductase) antioxidants, and xenobiotic-detoxifying enzymes (activities of glutathione S-transferase, NADPH:cytochrome P450 reductase, NADH:cytochrome b5 reductase). These effects were more severely expressed after the drought stress, suggesting that this cultivar is more tolerant to waterlogging than to drought stress.

The Physiological Responses of Wheat and Maize Seedlings Grown under Water Deficit Are Modulated by Pre-Application of Auxin-Type Plant Growth Regulators.

The physiological responses of wheat and maize seedlings to exogenous auxin-type compounds 1-[2-chloroethoxycarbonyl-methyl]-4-naphthalenesulfonic acid calcium salt (TA-12) and 1-[2-dimethylaminoethoxicarbonylmethyl]naphthalene chlormethylate (TA-14) application prior to polyethyleneglycol-6000 (PEG) treatment were studied. PEG treatment inhibited seedlings growth and caused alterations in their antioxidant defence which was crop-specific. PEG increased the non-enzymatic antioxidants along with inhibition of enzymatic antioxidant activity in wheat, while in maize the opposite effects were found. The TA-12 and TA-14 applied alone increased most of the growth parameters measured in both crops, as well as the catalase activity and protein content of wheat. The growth of PEG-treated wheat and maize plants was improved by foliar spray with TA-compounds (TAs). Application of TAs before PEG treatment maintained low-molecular weight thiol-containing compounds and protein contents, and catalase and peroxidase activities close to the control levels. This was better expressed in maize than in wheat seedlings. The results showed that the preliminary application of TA-12 and TA-14 can reduce the adverse effects of moderate water deficit by crop-specific adjustment of the antioxidant defence to counteract stress.

Modulation of Physiological Stress Response of Triticum aestivum L. to Glyphosate by Brassinosteroid Application.

The potential of brassinosteroids to modulate the physiological responses of winter wheat (Triticum aestivum L.) to herbicide stress was evaluated. Young winter wheat seedlings were treated with 24-epibrassinolide (EBL) and 24 h later were sprayed with glyphosate. The physiological responses of treated plants were assessed 14 days after herbicide application. Wheat growth was noticeably inhibited by glyphosate. The herbicide application significantly increased the content of the stress markers proline and malondialdehyde (MDA) evidencing oxidative damage. The content of phenolic compounds was decreased in the herbicide-treated plants. Slight activation of superoxide dismutase (SOD) and catalase (CAT) and considerable increase of glutathione reductase (GR) and guaiacol peroxidase (POX) activities were found. Increased POX and glutathione S-transferase (GST) activities were anticipated to be involved in herbicide detoxification. Conjugation with glutathione in herbicide-treated plants could explain the reduction of thiols suggesting unbalanced redox state. EBL application did not alter the plant growth but a moderate activation of antioxidant defense (POX, GR, and CAT activities and phenolic levels) and detoxifying enzyme GST was observed. The hormonal priming provoked a slight decrease in MDA and proline levels. The results demonstrate that EBL-pretreatment partly restored shoot growth and has a potential to mitigate the oxidative damages in glyphosate-treated plants through activation of the enzymatic antioxidant defense and increase of the phenolic compounds.

Physiological Responses of Wheat Seedlings to Soil Waterlogging Applied after Treatment with Selective Herbicide.

Waterlogging impairs crop development and considerably affects plant productivity worldwide. Wheat is sensitive to waterlogging. Serrate® (Syngenta) is a selective herbicide controlling annual grass and broadleaf weeds for use in wheat. To extend the existing information about the physiological effects of selective herbicides (Serrate® in particular) and subsequent waterlogging in wheat, we monitored phenotype alterations and examined key enzymatic and non-enzymatic antioxidant defense systems together with typical oxidative stress biomarkers. Seventeen-day-old wheat (Triticum asetivum L., cv. Sadovo-1) plants were sprayed with Serrate®; 72 h later, waterlogging was applied for 7 days, and then seedlings were left to recover for 96 h. The herbicide did not alter plant phenotype and increased antioxidant defense, along with H2O2 content, confirming the wheat's tolerance to Serrate®. Evident yellowing and wilting of the leaves were observed at 96 h of recovery in waterlogged wheat, which were stronger in plants subjected to Serrate® + waterlogging. Waterlogging alone and herbicide + waterlogging gradually enhanced the content of stress markers (malondialdehyde, proline, and H2O2), non-enzymatic antioxidants (low-molecular thiols and total phenolics), and the activity of superoxide dismutase, guaiacol peroxidase, and glutathione reductase. The effects of herbicide + waterlogging were stronger than those of waterlogging alone even during recovery, suggesting that Serrate® interacted synergistically with the subsequently applied flooding.

Assessment of the Biochemical Responses of Wheat Seedlings to Soil Drought after Application of Selective Herbicide.

Drought is a major environmental constrain with a deleterious effect on plant development leading to a considerable reduction of crop productivity worldwide. Wheat is a relatively drought tolerant crop during the vegetative stage. The herbicide Serrate® (Syngenta) is a preparation containing two active chemical substances with different modes of action, which inhibit the biosynthesis of fatty and amino acids. It is commonly used as a systemic and selective chemical agent to control annual grass and broadleaf weeds in cereal crops and particularly in wheat, which is tolerant to Serrate®. Seventeen-day-old wheat seedlings (Triticum aestivum L., cv. Sadovo-1) grown as soil culture under controlled conditions were sprayed with an aqueous solution of Serrate®. Seventy-two hours later the plantlets were subjected to drought stress for seven days to reach a severe water deficit followed by four days of recovery with a normal irrigation regime. Oxidative stress markers, non-enzymatic, and enzymatic antioxidants were analyzed in the leaves of plants from the different treatment groups (herbicide-treated, droughts-stressed, and individuals which were consecutively subjected to both treatments) at 0, 96, and 168 h of drought stress, and after 96 h of recovery. Herbicide treatment did not alter substantially the phenotype and growth parameters of the above-ground plant parts. It provoked a moderate increase in phenolics, thiol-containing compounds, catalase, superoxide dismutase, glutathione reductase, and H2O2. However, significant variations of malondialdehyde, proline, and peroxidase activity caused by the sole application of the herbicide were not detected during the experimental period. Drought and herbicide + drought treatments caused significant growth inhibition, increased oxidative stress markers, and activation of enzymatic and non-enzymatic antioxidant defense reaching the highest levels at 168 h of stress. Plant growth was restored after 96 h of recovery and the levels of the monitored biochemical parameters showed a substantial decline. The herbicide provoked an extra load of oxidative stress-related biochemical components which did not aggravate the phenotypic and growth traits of plants subjected to drought, since they exhibited a good physiological status upon recovery.

Long-term impact of sublethal atrazine perturbs the redox homeostasis in pea (Pisum sativum L.) plants.

Atrazine frequently contaminates soil, groundwater, rivers, and ponds. It is well know that acute doses (1-5 mM) of atrazine induce massive generation of singlet oxygen by blocking photosystem II. The sublethal concentrations of this herbicide, similar to those found in the environment, also reduce growth and disrupt photosynthesis in a long-term aspect, but exact mechanisms remain much uncertain. In this study the effects of environmentally relevant atrazine levels, ranging from 0.1 to 10 µM, on pea plants were characterized for up to 20 days. The plants exposed to continuous influence of atrazine exhibited perturbed redox homeostasis with increases of the lipid peroxides, the total and oxidized glutathione pools and elevated guaiacol peroxidase and glutathione-S-transferase activities. In contrast, the long-term atrazine impact did not affect superoxide dismutase activity whereas the catalase was inhibited. The perturbations of the redox status and the recruitment of the antioxidant machinery imply that the sublethal atrazine concentrations alter the poise between production and scavenging of reactive oxygen species. Taken together these results show that the long-term impact of sublethal atrazine has hallmarks of oxidative stress most probably triggered by generation of singlet oxygen.