Research Institute of Phytopathology

Moscow Region, Russia

Research Institute of Phytopathology

Moscow Region, Russia
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Baker C.J.,U.S. Department of Agriculture | Mock N.M.,U.S. Department of Agriculture | Aver'yanov A.A.,Research Institute of Phytopathology
Physiological and Molecular Plant Pathology | Year: 2017

Most methods used for the quantification of chlorogenic acids (CGA) in plant tissue use a multi-step organic solvent extraction, which improves accuracy but is laborious and time-consuming. We needed a simpler technique that would allow us to compare the effects of various bacterial treatments on leaf tissue CGA levels over time. Here we describe such a technique that was rapid, used less tissue, and was highly reproducible. The final technique involved grinding a small amounts of leaf tissue in acidified water, which helped stabilize the phenolics, and after clarification by centrifugation could directly be analyzed by Ultra High Performance Liquid Chromatography with Ultraviolet and Mass spectrophotometer detectors (UHPLC-UV-MS). Using this method, we found that CGA levels can vary greatly with leaf age, location on the leaf, and between plants. Therefore in order to observe the more subtle changes caused by bacterial treatments, it was essential to first be aware of the CGA variations in plants to find the most homogenous tissue to use. Ultimately, we found that leaf halves were most homogenous and adjacent panels on a same side of the midrib could be used for multiple sampling of a treatment over time. During this study we also found some interesting relationships regarding CGA distribution in the plant. © 2017


Baker C.J.,U.S. Department of Agriculture | Mock N.M.,U.S. Department of Agriculture | Orlandi E.W.,Pennsylvania State University | Deahl K.L.,U.S. Department of Agriculture | And 4 more authors.
Physiological and Molecular Plant Pathology | Year: 2011

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2-4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR-), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili. © 2011 Elsevier Ltd.


Baker C.J.,U.S. Department of Agriculture | Owens R.A.,U.S. Department of Agriculture | Whitaker B.D.,U.S. Department of Agriculture | Mock N.M.,U.S. Department of Agriculture | And 3 more authors.
Physiological and Molecular Plant Pathology | Year: 2010

Plants are capable of producing a wide array of secondary metabolites that serve a variety of functions, due to their bioactive, redox or structural properties. Subtle changes in the external or internal environment of the plant can cause significant changes in the array of secondary metabolites present in the tissue. During the last 10 years the critical roles that these metabolites play both in plant development as well as plant defense against biological and abiotic stresses have begun to become more widely appreciated. Here we describe changes in apoplastic phenolics associated with systemic infections of Potato spindle tuber viroid (PSTVd) as it spreads through inoculated tomato plants. Apoplast fluids from leaves at different distances from the infection site were examined by HPLC-UV over a 5 week period. The phenolic composition of apoplasts in healthy plants was highly dynamic and depended on leaf position as well as plant age. Differences in the apoplast composition of PSTVd infected plants were detected only in leaves in which the viroid was present and multiplied. In those leaves, apoplast phenolics that were similar to the control generally decreased although some increased compared to control plants. Another set of phenolics that were unique to the viroid infected plants appeared simultaneously with the onset of viroid replication and then disappeared. An important basic finding of this study is that the composition of apoplastic metabolites is dynamic, changing with time and leaf development. In addition we demonstrate that the secondary metabolites of the leaf apoplast respond to the presence of the viroid in the symplast of the cell. This study provides a basis for a more detailed investigation of the dynamics and identification of the apoplastic phenolics involved in the viroid-host interaction. © 2010.


Baker C.J.,U.S. Department of Agriculture | Mock N.M.,U.S. Department of Agriculture | Whitaker B.D.,U.S. Department of Agriculture | Hammond R.W.,U.S. Department of Agriculture | And 3 more authors.
Physiological and Molecular Plant Pathology | Year: 2014

Previous work demonstrated that when tobacco cell suspensions were inoculated with certain bacterial strains, the redox potential of the suspensions would increase (oxidative), as much as 100mV, and in some cases last more than an hour. To discover possible contributors to this prolonged redox potential burst, we examined the oxidation of various plant phenolics that were similar to those found in the cell suspensions. Acetosyringone, one of the major extracellular phenolics in tobacco cell suspensions, was unique in causing a prolonged increase in redox potential of more than 100mV when incubated with H2O2 and peroxidase. The increase in potential appears to be due to a relatively stable radical intermediate. The length of time that the redox potential increased was dependent on the ratio of H2O2 and acetosyringone. The most favorable was a 1:1 ratio. Invitro characterization showed that production of this intermediate is favored by relatively low pH (around 6) and cooler temperatures possibly due to slower kinetics. Phosphate buffer was substituted for MES buffer, which had been used initially but was found to interfere with the oxidation. The results of this study demonstrate that apoplastic phenolics, in addition to often playing a role as antioxidants that reduce H2O2, can also cause rapid transient periods of extreme redox potential creating a local environment hostile to pathogen ingress. © 2014.


Mock N.M.,U.S. Department of Agriculture | Baker C.J.,U.S. Department of Agriculture | Aver'yanov A.A.,Research Institute of Phytopathology
Physiological and Molecular Plant Pathology | Year: 2015

Acetosyringone is a phenolic metabolite often found in plant apoplasts. Its oxidation by hydrogen peroxide and peroxidase results in a prolonged increase in the redox potential of the reaction mixture, similar to redox increases observed in tobacco suspension cells upon treatment with incompatible bacteria. Since high redox potentials, being oxidative, are generally detrimental to bacteria, the effect of acetosyringone oxidation on bacterial viability was examined. Pseudomonas syringae pv. syringae was added to reaction mixtures containing acetosyringone, hydrogen peroxide and peroxidase and samples were removed to determine viability by dilution plating. Initial studies were done with low bacterial concentrations, 105CFUml-1, to ensure that scavenging of H2O2 was negligible and did not interfere with the reaction mixture. No colonies were formed by bacteria that had been added to reaction mixtures with acetosyringone ranging from 25 to 100μ. Examination of the bacteria by microscopy and flow cytometry, using fluorescent stains that indicate bacterial membrane integrity, suggested that these bacteria had maintained their membrane integrity. In addition they were able to respire based on oxygen uptake. When bacteria were added to on-going reaction mixtures at a time point after the prolonged redox response, the CFUml-1 increased indicating that a stable reaction product was not responsible for the non-culturability bioactive effect. Other bacterial isolates, P.s. pv. tabaci and Pseudomonas fluorescens, were less susceptible to the bioactive effect of the acetosyringone oxidation. Other phenolics were tested and had lesser degrees of bioactivity and in some cases reduced the bioactivity of acetosyringone oxidation. The 'viable but non-culturable' (VBNC) state of the bacteria in this study is compared to that described for other medical and plant pathogens. © 2014.


Baker C.J.,U.S. Department of Agriculture | Kovalskaya N.Y.,Pennsylvania State University | Mock N.M.,U.S. Department of Agriculture | Owens R.A.,U.S. Department of Agriculture | And 5 more authors.
Physiological and Molecular Plant Pathology | Year: 2012

The plant leaf apoplast is one of the first lines of defense against many foliar pathogens. The aqueous layer lining the airspace within leaves is enriched with secondary metabolites that can serve many roles including protection against environmental hazards, both biotic and abiotic. The constituents and their concentration change as the leaf matures or undergoes stress. To monitor and quantify changes in these metabolites during pathogen stress, we needed a more sensitive technique. We were able to modify the infiltration-centrifugation technique to use smaller samples, individual tomato leaflets, plus use an internal standard which indicated the amount of dilution in each sample. Dinotefuran, a neonicotinoid used as a systemic pesticide, proved to be resistant to the redox environment of the apoplast and had similar chemical properties allowing it to be analyzed under the same UPLC conditions as the other phenolic metabolites. Water soluble metabolites on the leaf surface were found to be a major source of contamination that could be avoided by rinsing leaves with water prior to infiltration. The improved sample efficiency and accuracy provided by this technique, along with the use of Dinotefuran as an internal standard, will provide the sensitivity needed to monitor apoplast metabolites during pathogen stress. © 2012.


Zakharenkova T.S.,Research Institute of Phytopathology | Aver'yanov A.A.,Research Institute of Phytopathology | Pasechnik T.D.,Research Institute of Phytopathology | Lapikova V.P.,Research Institute of Phytopathology | Baker C.J.,U.S. Department of Agriculture
Physiological and Molecular Plant Pathology | Year: 2012

Small amounts of water placed onto leaf surface for one day just before challenge inoculation were found to reduce severity of blast disease of rice and cucurbit scab of cucumber. The effect was only local in the first pathosystem and both local and systemic in the second one. In rice, the reduction in symptoms was preceded by increased superoxide production in the treated leaves. Presumably, water liberates plant elicitors inducing oxidative burst and other defense responses that render the plant relatively resistant. This reaction may be an adaptation of plants to higher risk of infections under humid conditions. © 2012 Elsevier Ltd.


Baker C.J.,U.S. Department of Agriculture | Kovalskaya N.Y.,U.S. Department of Agriculture | Mock N.M.,U.S. Department of Agriculture | Deahl K.L.,U.S. Department of Agriculture | And 4 more authors.
Physiological and Molecular Plant Pathology | Year: 2013

In both plants and animals, there has been a strong focus on reactive oxygen species and antioxidants in regard to stress responses. This has led to an awareness of the importance of 'redox status' as a prime regulatory determinant of cellular function and responses to internal and external stimuli. It has been difficult to study the effect of or fluctuation in the redox potential during interactions, since the frequency of sampling is limiting. We tested a method of using redox electrodes with bacterial and plant cell suspensions to monitor the extracellular redox potential during different plant/bacterial interactions. The advantage of the electrodes is that they provide continuous and nonintrusive monitoring of the redox potential, and interact with a larger array of metabolites. We found four different responses of redox potential to different plant/bacterial interactions. The redox responses are coincident with phenolic changes in the extracellular fluid of the suspensions. Further investigation of the mechanisms and parameters that affect the technique will provide insight into using electrodes to measure redox potential in planta. © 2013.


Aver'yanov A.A.,Research Institute of Phytopathology | Lapikova V.P.,Research Institute of Phytopathology | Pasechnik T.D.,Research Institute of Phytopathology | Abramova O.S.,Peoples' Friendship University of Russia | And 3 more authors.
Fungal Biology | Year: 2014

Many environmental factors, alone or combined, affect organisms by changing a pro-/antioxidant balance. Here we tested rice blast fungus (Magnaporthe oryzae) for possible cross-adaptations caused by relatively intense light and protecting from artificially formed reactive oxygen species (ROS) and ROS-dependent fungitoxic response of the host plant. Spore germination was found to be suppressed under 4-hand, to larger extent, 5-hillumination. The effect was diminished by antioxidants and, therefore, suggests involvement of ROS. One-hour of light did not affect spore germination, but stimulated their chemically assayed superoxide production. The illuminated spores were more tolerant (than non-illuminated ones) to artificially generated H2O2, O2-, or OH or to toxic diffusate of rice leaf. They also caused more severe disease symptoms if applied to leaves of the susceptible rice cultivar at low concentration. Spore diffusates decomposed hydrogen peroxide. They detoxified exogenous H2O2 and superoxide radical as well as leaf diffusates. Spore illumination increased some of these protective effects. It is suggested that short-term light led to mild oxidative stress, which induced spore antioxidant capacity, enhancing spore tolerance to subsequent stronger oxidative stress and its aggressiveness in planta. Such tolerance depends partly on the antidotal action of spore extracellular compounds, which may also be light-stimulated. Therefore, a certain ROS-related environmental factor may adapt a fungus to other factors and so modulate its pathogenic properties. © 2014 The British Mycological Society.


PubMed | Research Institute of Phytopathology, Peoples' Friendship University of Russia and U.S. Department of Agriculture
Type: Journal Article | Journal: Fungal biology | Year: 2014

Many environmental factors, alone or combined, affect organisms by changing a pro-/antioxidant balance. Here we tested rice blast fungus (Magnaporthe oryzae) for possible cross-adaptations caused by relatively intense light and protecting from artificially formed reactive oxygen species (ROS) and ROS-dependent fungitoxic response of the host plant. Spore germination was found to be suppressed under 4-h and, to larger extent, 5-h illumination. The effect was diminished by antioxidants and, therefore, suggests involvement of ROS. One-hour of light did not affect spore germination, but stimulated their chemically assayed superoxide production. The illuminated spores were more tolerant (than non-illuminated ones) to artificially generated H(2)O(2), O(2)(-), or OH or to toxic diffusate of rice leaf. They also caused more severe disease symptoms if applied to leaves of the susceptible rice cultivar at low concentration. Spore diffusates decomposed hydrogen peroxide. They detoxified exogenous H(2)O(2) and superoxide radical as well as leaf diffusates. Spore illumination increased some of these protective effects. It is suggested that short-term light led to mild oxidative stress, which induced spore antioxidant capacity, enhancing spore tolerance to subsequent stronger oxidative stress and its aggressiveness in planta. Such tolerance depends partly on the antidotal action of spore extracellular compounds, which may also be light-stimulated. Therefore, a certain ROS-related environmental factor may adapt a fungus to other factors and so modulate its pathogenic properties.

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