The aim of the work was to shed light into histological, physiological and molecular changes of Fagus sylvatica seedlings infected with the root pathogen Phytophthora citricola with the final goal to distinguish between local and systemic responses. Real-time quantitative PCR analysis proved that P. citricola was able to grow from infected roots into hypocotyl and epicotyl tissue of F. sylvatica seedlings. Light microscopy showed many collapsed parenchyma cells of the cortex without being penetrated by the pathogen. Hyphae were mainly growing intracellular in parenchyma and xylem tissue. Transmission electron microscopy displayed disintegration of xylem vessels and of parenchyma cells. Inhibition of water uptake of infected beech seedlings was positively correlated with the concentration of zoospores used in the experiment. In addition, a split root experiment indicated that invertases were possibly involved locally and systemically in the conversion of sucrose of P. citricola infected roots. During the growth of the pathogen in roots, a transient expression of the 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase gene was quantified in leaves which was detected in parallel with the first peak of a biphasic ethylene outburst. Additionally a systemic upregulation of aquaporin transcripts was mainly detected in leaves of beech seedlings infected with P. citricola .

The growth-differentiation balance hypothesis (GDBH) predicts changes in susceptibility of plants against herbivores with changing resource availability. In the presented study we tested the validity of the GDBH for trees infected with a root pathogen. For this purpose Fagus sylvatica seedlings grown under different atmospheric CO2- and soil nitrogen regimes were infected with the root pathogen Phytophthora citricola. High nitrogen supply increased total biomass of beech regardless of the CO2-treatment, whereas elevated CO2 enhanced biomass only in the high nitrogen treatment. The responses of beech under the different growing regimes to the Phytophthora root infection were not in line with the predictions of the GDBH. Enhanced susceptibility of beech against P. citricola was found in seedlings grown under elevated CO2 and low nitrogen supply. Fifteen months after inoculation these plants were characterized by enhanced water use efficiency, by altered root–shoot ratios, and by enhanced specific root tip densities.

In several studies of oak decline in Europe, a semi-papillate homothallic Phytophthora taxon was consistently isolated, together with other Phytophthora species, from rhizosphere soil samples. It was also found associated with necrotic fine roots and stem necroses of Fagus sylvatica and Alnus glutinosa. Due to morphological and physiological similarities, the semi-papillate isolates were previously identified as P. syringae by various authors. The morphology, physiology and pathogenicity against fine roots of Quercus robur, Q. petraea and F. sylvatica, bark of A. glutinosa, leaves of flex aquifolium and apple fruits of this Phytophthora species are described and compared with those of related and similar Phytophthora species, namely P. ilicis, P. psychrophila, P. quercina, P. citricola and P. syringae. The phylogenetic placement on the basis of ITS and mtDNA sequence data was also examined. Isolates of this taxon produce colonies with stellate to rosaceous growth patterns and limited aerial mycelium on various agar media. Antheridia are predominantly paragynous. In water culture catenulate hyphal swellings and semi-papillate caducous sporangia, that are usually limoniform, ellipsoid or ovoid, are formed abundandly, mostly in lax or dense sympodia. This taxon is a moderately slow growing, low temperature species with optimum and maximum temperatures around 20 and 25 degreesC, respectively. Tested isolates are moderately aggressive to fine roots of oaks and beech, highly aggressive to holly leaves and apple fruits, and slightly pathogenic to alder bark. Thirteen tested isolates had an identical and distinct ITS sequence which was more similar to that of P. ilicis and P. psychrophila than any other known taxa. On the basis of their unique combination of morphological characters, colony growth patterns, cardinal temperatures for growth, growth rates, pathogenicity to oaks, beech, alder, apple and holly, their host range, and ITS and mtDNA sequences the semi-papillate caducous Phytophthora isolates from oaks, beech and alder are clearly separated from related and similar Phytophthora spp., and described as a new species, P. pseudosyringae sp. nov.

Extensive investigations on the fine root status of declining and healthy spruce were conducted in several stands at higher elevations of the Bavarian Forest heavily affected by needle yellowing. In most of the root parameters recorded, yellowing trees had significantly lower values than neighbouring green trees. Tight correlations were found between decreasing fine root density and crown transparency, degree of yellowing ( increasing) and needle Mg( Ca) contents ( decreasing), respectively. Although growing on the same substrate, green trees showed much better Mg ( Ca) nutrition than yellow trees, indicating that poor fine root status contributes to Mg ( Ca) deficiency in yellowing spruce. Experiments with spruce seedlings growing in soil samples from yellowing stands proved that needle symptoms can easily be reproduced on the seedlings under controlled conditions (i.e. in the absence of adverse atmospheric factors). Furthermore, reduced fine root systems and severe root damage were observed on seedlings grown in soils from yellowing stands, but not on those in soils from green stands. Adding a layer of soil from a yellow stand to a soil from a green stand caused a decrease of root parameters. Needle as well as root symptoms in these experiments were largely ameliorated after soils had been heat (autoclaving, sterilisation) or fungicide treated. Plants from treated soils had significantly longer roots and more root tips. The results of our study indicate that Mg deficiency leading to severe needle yellowing in stands at higher elevations of the Bavarian Forest is at least partially mediated by fine root disorders. Also, strong evidence is presented that fine root damage on trees in the affected stands is caused by soilborne micro-organisms, most likely fungi. Their exact identity, however, still remains to be unravelled.

In this contribution, we compare the influence of four different Phytophthora species on root development, net CO2 assimilation and transpiration of beech seedlings and saplings. Some few days after inoculation, photosynthesis and transpiration of seedlings infected with either P. citricola or P. cambivora were strongly reduced. In parallel, about 60% of their root systems was destroyed compared to control plants. Three weeks after infection, all seedlings were dead, showing severe wilt symptoms on leaves. Remarkably, P. syringae and P. undulata infected seedlings and older beech plants did not differ from controls regarding photosynthesis and transpiration, although the root systems were damaged. However, a significant influence on net CO2 assimilation and transpiration of P. citricola infected beech saplings was visible after bud break in the following year. Some days before plants started to wilt, photosynthesis and transpiration were reduced to almost zero. Water use efficiency data (WUE) clearly indicated that infected plants suffered from severe drought.

We report on the growth pattern of the root rot pathogen Phytophthora quercina in roots of oak seedlings and on the localization of its elicitin quercinin. Some hours after infection, the pathogen was mainly found in the intercellular spaces of the cortical parenchyma. Some parenchyma cells were also invaded by P. quercina and the plasmalemma of those cells as well as of neighbouring cells separated from the cell wall. Transmission electron microscopy studies proved that P. quercina penetrated the cell walls forming haustorial-like structures in invaded cells. These structures and cell wall appositions were covered with electron dense material. Immunofluorescence investigations in combination with a specific anti cryptogein antibody clearly showed that the P. quercina elicitin quercinin was located within the hyphal cell wall and seems to be released into invaded cells. In addition, immunogold labelling showed that quercinin was found in the intercellular spaces as well as in penetrated cells. Finally, we demonstrated with ELISA that quercinin was produced by the pathogen in infected tissue during the whole growth phase.

A description is given of the use of a combination of polymerase chain reaction (PCR) and baiting techniques for the specific detection of Phytophthora quercina and Phytophthora citricola from soil around declining oak trees. The soil was flooded with water and subjected to a specific baiting procedure using Quercus robur leaflets as baits. Single round or nested PCR, respectively, with species‐specific primers allowed the detection of P. quercina and P. citricola in infected oak leaflets used as baits and in the water from the same bait tests. PCR detection of both fungi was also possible after soil samples had been thoroughly mixed with water and the floating organic debris had been collected. Phytophthora quercina and P. citricola could be readily detected in almost every case in the water from these tests by PCR but less frequently in the organic debris. The identities of P. quercina and P. citricola were confirmed by restriction digests of the corresponding PCR amplicons. The presence of both fungi was also confirmed in parallel in soil samples tested by baiting with oak leaflets. Nested PCR with the primers used allowed the detection of as few as five zoospores of P. citricola and 300 zoospores of P. quercina in a volume of 100 μ l. The methods presented here allow detection and identification of species of Phytophthora in soil without the need for direct extraction of soil samples, and without specific knowledge of the morphological characteristics of the genus.

Naphthazarin toxins such as dihydrofusarubin, marticin, isomarticin and methyljavanicin are produced by Fusarium solani, a pathogen for citrus, peas and tomatoes. Similar to other p-quinones such as several members of the anthraquinone- or 1,4-naphthoquinone-families, dihydrofusarubin, marticin, and isomarticin were reductively activated by diaphorases at the expense of NAD(P)H. Upon aut:oxidation, the reduced compounds activated oxygen, forming superoxide and hydrogen peroxide. Finally, a strong oxidant similar to the OH .-radical was produced in an iron-dependent: reaction. The Fusarium solani toxins did not show redox-coupling with isolated mitochondria neither at the expense of NADH nor of succinate. However, the naphthazarin derivates marticin, isomarticin and methyljavanicin, but not dihydrofusarubin, acted as uncouplers of the mitochondrial electron transport chain.

Dihydrofusarubin, marticin and isomarticin are naphthazarin toxins produced by Fusarium solani, a pathogen of citrus, peas and tomato. Like other p-quinones such as 1,4-naphthoquinone, menadione or juglone, these compounds can be reductively activated by dithiols such as dihydrolipoic acid (reduced thioctic acid) or dithiothreitol but not by monothiols such as cystein or glutathione. Upon autoxidation, reduced p-quinones with appropriately negative redox potentials between −100 and −250 mV activate oxygen in a dithiol-dependent reaction producing superoxide, hydrogen peroxide and a strong oxidant similar to the OH-radical-type oxidant («crypto-OH»), but apparently not identical to the free OH-radical.

In the presence of riboflavin benzylaminopurine (BAP) induces ethane formation from a-linolenic acid in the light. Addition of Cu2+-ions leads to the formation of ethylene at the expense of ethane. Ethylene release from α-linolenic acid starts with copper ions at concentrations between 0.1 and μM. Similar results are described for rose bengal as a further photodynamic active compound. On the basis of these results we present model reactions for a light-dependent ethylene formation from α-linolenic acid catalyzed by cytokinin derivatives and copper ions.

The effects of the Fusarium solani toxin dihydrofusarubin on tobacco leaves and on spinach chloroplasts have been studied in comparison with the herbicide methylviologen. Toxin treatment of tobacco leaves and leaf discs resulted in a light dependent chlorophyll degradation and a simultaneous ethane production. Experiments with spinach chloroplasts showed that dihydrofusarubin interacted with the electron transport chain, thus forming reactive oxygen species. Experiments with DCMU and DBMIB proved that superoxide was produced on photosystem I in a light dependent reaction. Furthermore, we showed that NADPH formation was blocked by dihydrofusarubin. Inhibition started with 10 µmol/L DHF, whereas about 80% inhibition of NADP+-reduction was achieved with 100 µmol/L toxin. Inhibition of NADPH-formation was accompanied by a simultaneous increase in ATP-production. Experiments with methylviologen proved that the effects of the Fusarium solani toxin dihydrofusarubin on chloroplasts were comparable with the effects of this herbicide. The significance of the dihydrofusarubin toxin for symptom development on citrus trees infected with Fusarium solani is discussed.

After hydrolysis of chitin from fungal cell walls, the glucosamine released was derivatized with phenylisothiocyanate and was used to quantify fungal infection of different plant tissues. The glucosamine-phenylisothiocyanate derivative (GlcN-PiTC) was separated on a reversed-phase HPLC column and quantified afterwards using GlcN-PiTC standards. The advantage of this method is its sensitivity and high reproducibility in a wide range of concentrations.

The influence of cinnamic acid derivatives on the H2O2-independent oxidation of indole acetic acid by horseradish peroxidase was examined. Cinnamic acid derivatives show a sharp increase from a slight stimulation of the oxidative reaction to a complete inhibition in a very narrow concentration range. This threshold effect occurs not only for the diphenols caffeic and dihydrocaffeic acids but also for the monophenols ferulic and sinapic acids. The latter phenols are not demethylated to caffeic acid by horseradish peroxidase during the reaction. They are both inhibitors on their own account. We, therefore, conclude that the inhibition is not only due to the radical scavenging properties of the inhibitors; direct interactions between the inhibitors and peroxidase also play a role in the unusual behaviour of the inhibitors.

SynopsisGreen plants, within certain limitations, can adapt to a wide variety of unfavourable conditions such as drought, temperature changes, light variations, infectious attacks, air pollution and soil contamination. Depending on the strength of the individual impact(s), fluent or abrupt changes in visible or measurable stress symptoms indicate the deviation from normal metabolic conditions. Most of the visible or measurable symptoms are connected with altered oxygen metabolism principally concerning the transition from mostly heterolytic (two-electron transition) to increased homolytic (one-electron transition) processes. Homolytic reactions within metabolic sequences create, however, free radicals and have to be counteracted by the increase in radical-scavenging processes or compounds, thus warranting reaction sequences under metabolic control. At later states of stress episodes, the above control is gradually lost and more or less chaotic radical processes take over. Finally, cellular decompartmentalisations induce lytic and necrotic processes which are visible as the collapse of darkening cells or tissues. Every episode during this process is governed by a more or less denned balance between pro- and antioxidative capacities, including photosynthetic (strongly under metabolic and oxygen-detoxifying control) and photodynamic (only controlled by scavenger- and/or quencher-availability) reactions. This (theoretical) sequence of events in most cases can only be characterised punctually by strongly defined (analytical) indicator reactions (ESR) and is certainly species- and organ-specific.

The stability behaviour and the recovery rates of spruce needle enzymes such as superoxide dismutase, dehydroascorbic acid reductase, glutathione reductase, catalase and peroxidase were determined. Also the isoelectric points were measured for catalase to be 8.0, for peroxidase to be 6.0 and 9.4 and for glutathione reductase to be 4.6. Furthermore, the activities of superoxide dismutase, dehydroascorbic acid reductase, glutathione reductase, catalase and peroxidase were compared for healthy and damaged spruce needles. A significant increase in enzymatic activities was measured for dehydroascorbic acid reductase glutathione reductase and catalase of damaged compared to healthy needle segments. However, no difference in enzymatic activities was found for superoxide dismutase and peroxidase of healthy and damaged needle samples.

The ice nucleation activity of Pseudomonas syringae Cit 7 bacteria in aqueous suspensions or on citrus leaves was determined. Pretreatment of the bacteria with TCDO and hemine in Kings Medium B had no effect on their ice nucleation activity (INA). However, bacterial growth was blocked. When Kings Medium was substituted for by phosphate buffer (0.2 mol/l, pH = 7.0), preincubation of Pseudomonas syringae with TCDO and hemine resulted in a complete loss of INA. Additionally, all bacteria were killed. Both INA and viability of bacteria on citrus leaves were also lost upon treatment with TCDO and hemine. The effect of TCDO and hemine on the bacteria strongly depended on the time of preincubation of the two compounds.

Three-year-old clonal Picea abies (L.) Karst. plants, grown either on a sandy (No. 1) or on a calcareous (No. 2) soil, were treated with ozone (100 μg m−3 and peaks of up to 360 μg m−3) and acid mist (pH 3·0) over two vegetation periods. Needles of the current (1987) and previous (1986) year were analysed at the end of the experiment for biosynthetic enzymes and in vivo activity of the phenylpropanoid pathway, for products of polyphenol metabolism (clones 11 and 14), and for mono- and sesquiterpenes (clones 14 and 16). 1.1. Polyphenol metabolism. The activity of cinnamyl alcohol dehydrogenase, an enzyme of the lignin pathway, was increased by the treatment by up to 83% in the needles of both age classes. Chalcone synthase was measured in mature tree material for the first time. This enzyme, as well as phenylalanine ammonia-lyase activities and the content of catechin, astringin, isorhapontin, picein and p-hydroxyacetophenone, exhibited no significant treatment-dependent differences. However, soil and age-class dependent differences occurred. Pulse-labelling experiments with l[U-14C]phenylalanine and [2-14C]acetate were carried out at four different stages of shoot development and showed label incorporation into (+)-catechin and proanthocyanidins. There was no effect of the ozone and acid mist treatment.2.2. Monoterpenes. The content of needle terpenes was generally diminished by the ozone and acid mist treatment. Other factors tested, such as clone, needle age and soil, exerted a highly significant influence on the content of most of the needle monoterpenes.

Geschädigte Nadeln zeigen im Vergleich zu grünen Nadeln erhöhte Gehalte an Ascorbinsäure und Glutathion. Die Photosyntheseprodukte Glucose, Fructose, Saccharose und Stärke sind in gelben Nadeln deutlich erniedrigt. Vergilbte Nadeln sind durch niedrige Kaliumgehalte charakterisiert, wogegen die Magnesium, Calcium- und Mangangehalte über denen grüner Nadeln liegen.

Aqueous leaf extracts from Dionaea muscipula contain quinones such as the naphthoquinone plumbagin that couple to different NADH-dependent diaphorases, producing superoxide and hydrogen peroxide upon autoxidation. Upon preincubation of Dionaea extracts with certain diaphorases and NADH in the presence of serumalbumin (SA), subsequent tryptic digestion of SA is facilitated. Since the secretroy glands of Droseracea contain proteases and possibly other degradative enzymes it is suggested that the presence of oxygen-activating redox cofactors in the extracts function as extracellular predigestive oxidants which render membrane-bound proteins of the prey (insects) more susceptible to proteolytic attacks.