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Inflorescences vs leaves: A distinct modulation of carbon metabolism process during Botrytis infection
Abstract
Plant growth and survival depends critically on photo assimilates. Pathogen infection leads to changes in carbohydrate metabolism of plants. In this study, we monitored changes in the carbohydrate metabolism in the grapevine inflorescence and leaves using Botrytis cinerea and Botrytis pseudo cinerea. Fluctuations in gas exchange were correlated with variations in chlorophyll a fluorescence. During infection, the inflorescences showed an increase in net photosynthesis with a stomatal limitation. In leaves, photosynthesis decreased, with a non-stomatal limitation. A decrease in the effective quantum yield (ФPSII) was accompanied by an increase in photochemical (qP) and non-photochemical quenching (qN). The enhancement of qP and ФPSII could explain the observed increase in Pn. In leaves, the significant decline in ФPSII and qP, and increase in qN suggest that energy was mostly oriented towards heat dissipation instead of CO2 fixation. The accumulation of glucose and sucrose in inflorescences and glucose and fructose in the leaves during infection indicate that the plant's carbon metabolism is differently regulated in these two organs. While a strong accumulation of starch was observed at 24 and 48 hpi with both species of Botrytis in the inflorescences, a significant decrease with B. cinerea at 24 hpi and a significant increase with Botrytis pseudo cinerea at 48 hpi were observed in the leaves. Based on these results, it can be said that during pathogen attack, the metabolism of grapevine inflorescence and leaf is modified suggesting distinct mechanisms modifying gas exchange, PSII activity and sugar contents in these two organs.
... al., 2014;Kumari et al., 2014;Kuzmanovska et al., 2012;Leyronas et al., 2014;Lorenzini & Zapparoli, 2014;Ma & Michailides, 2005;Makris et al., 2022;Martinez et al., 2003;Mercier et al., 2019;Moparthi et al., 2023;Muñoz & Campos, 2013;Muñoz et al., 2002Muñoz et al., , 2016Naegele et al., 2021;Pei et al., 2019;Polat et al., 2018;Rajaguru & Shaw, 2010;Román Ramos, 2013;Tanović et al., 2009Tanović et al., , 2014Testempasis et al., 2020;Toffolatti et al., 2020;Topolovec-Pintarić et al., 2004;Törün & Biyik, 2022;Vaczy et al., 2008;Vatsa-Portugal et al., 2014;Vercesi et al., 2014;Walker, 2013;Wessels et al., 2013Wessels et al., , 2016Zhang et al., 2018). The metadata is accessible on Dryad https:// doi. ...
Botrytis cinerea Pers. Fr. (teleomorph: Botryotinia fuckeliana ) is a necrotrophic fungal pathogen that attacks a wide range of plants. This updated pathogen profile explores the extensive genetic diversity of B. cinerea , highlights the progress in genome sequencing, and provides current knowledge of genetic and molecular mechanisms employed by the fungus to attack its hosts. In addition, we also discuss recent innovative strategies to combat B. cinerea .
Taxonomy
Kingdom: Fungi, phylum: Ascomycota, subphylum: Pezizomycotina, class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae, genus: Botrytis , species: cinerea .
Host range
B. cinerea infects almost all of the plant groups (angiosperms, gymnosperms, pteridophytes, and bryophytes). To date, 1606 plant species have been identified as hosts of B. cinerea .
Genetic diversity
This polyphagous necrotroph has extensive genetic diversity at all population levels shaped by climate, geography, and plant host variation.
Pathogenicity
Genetic architecture of virulence and host specificity is polygenic using multiple weapons to target hosts, including secretory proteins, complex signal transduction pathways, metabolites, and mobile small RNA.
Disease control strategies
Efforts to control B. cinerea , being a high‐diversity generalist pathogen, are complicated. However, integrated disease management strategies that combine cultural practices, chemical and biological controls, and the use of appropriate crop varieties will lessen yield losses. Recently, studies conducted worldwide have explored the potential of small RNA as an efficient and environmentally friendly approach for combating grey mould. However, additional research is necessary, especially on risk assessment and regulatory frameworks, to fully harness the potential of this technology.
... Cell wall transformation hydrolyzes sucrose into glucose and fructose and then introduces hexose into the target cells [45,46]. Transcriptomic analysis of A. thaliana and P. brassicae showed that sucrose synthase (SUS) and sugar permeases were induced during gall formation of P. brassicae, and cytoplasmic invertase mutant cinv1,2 had resistance to clubroot disease at 26 days post-inoculation (DPI), indicating that P. brassicae formed a new sink [47]. Soluble sugar accumulates in large amounts in infected organs during pathogen infection, and it has been proposed that sucrose and hexose accumulation in the apoplast is caused by enhanced sugar efflux from host cells [2,48]. ...
Plasmodiophora brassicae, an obligate intracellular pathogen, can hijack the host’s carbohydrates for survival. When the host plant is infected by P. brassicae, a large amount of soluble sugar accumulates in the roots, especially glucose, which probably facilitates the development of this pathogen. Although a complete glycolytic and tricarboxylic acid cycle (TCA) cycle existed in P. brassicae, very little information about the hexose transport system has been reported. In this study, we screened 17 putative sugar transporters based on information about their typical domains. The structure of these transporters showed a lot of variation compared with that of other organisms, especially the number of transmembrane helices (TMHs). Phylogenetic analysis indicated that these sugar transporters were far from the evolutionary relationship of other organisms and were unique in P. brassicae. The hexose transport activity assay indicated that eight transporters transported glucose or fructose and could restore the growth of yeast strain EBY.VW4000, which was deficient in hexose transport. The expression level of these glucose transporters was significantly upregulated at the late inoculation time when resting spores and galls were developing and a large amount of energy was needed. Our study provides new insights into the mechanism of P. brassicae survival in host cells by hijacking and utilizing the carbohydrates of the host.
... defenses (Vatsa-Portugal et al., 2015). Therefore, the photosynthate accumulation induced by infection with P. fluorescens ALEB7B is preferentially used by A. lancea to synthesize volatile oils that have antimicrobial effects (Fig. S2B) (Wang et al., 2009). ...
Atractylodes lancea is a well-known, but endangered, Chinese medicinal plant whose volatile oils are its main active components. As the volatile oil content in cultivated A. lancea is much lower than that in the wild herb, the application of microbes or related elicitors to promote growth and volatile oil accumulation in the cultivated herb is an important area of research. This study demonstrates that the endophytic bacterium Pseudomonas fluorescens ALEB7B isolated from the geo-authentic A. lancea can release several nitrogenous volatiles, such as formamide and N,N-dimethyl-formamide, which significantly promote the growth of non-infected A. lancea. Moreover, the main bacterial volatile benzaldehyde significantly promotes volatile oil accumulation in non-infected A. lancea via activating plant defense responses. Notably, the bacterial nitrogenous volatiles cannot be detected in the A. lancea - P. fluorescens symbiont while the benzaldehyde can be detected, indicating the nitrogenous volatiles or their precursors may have been consumed by the host plant. This study firstly demonstrates that the interaction between plant and endophytic bacterium is not limited to the commonly known physical contact, extending the ecological functions of endophyte in the phytosphere and deepening the understandings about the symbiotic interaction.
Six-year-old Vitis vinifera L. Chardonnay grapevines grown under controlled conditions were investigated with regard to both qualitative and quantitative temporal evolution of the xylem sap composition. Xylem exudates were collected by pruning the spurs just before bud burst. Potassium was the main mineral ion found in the sap (7300 µM), followed by calcium (2600 µM), phosphate (2500 µM), nitrate (2300 µM), sulfate (1600 µM), magnesium (500 µM), and chloride (150 µM). The major organic compounds were amino acids (5500 µM) and amides (mainly glutamine, 4000 µM), organic acids (3500 µM, mainly malate), and soluble carbohydrates (500 µM) including glucose, fructose, and sucrose. Over a one-week time course, water flux increased from the first four hours of exudation until 52 hours and then diminished steadily. Mineral ion concentrations remained fairly constant from the onset of the exudation to the end of the collection period, except for potassium which increased continuously and nitrate whose concentration reached a plateau after 24 hours. The high xylem levels of soluble organic compounds (amino acids and soluble carbohydrates), that were found initially, dropped rapidly and then increased reaching a maximum at 52 hours. Finally, the concentration decreased until the end of the exudation period. Furthermore, attention was paid to the effect on the xylem sap composition of recutting the spurs. The data obtained revealed that water flux and soluble carbohydrate release were enhanced, as a result of the recut, up to the maximum level previously reached at 52 hours.
Six-year-old Vitis vinifera L. Chardonnay grapevines grown under controlled conditions were investigated with regard to both qualitative and quantitative temporal evolution of the xylem sap composition. Xylem exudates were collected by pruning the spurs just before bud burst. Potassium was the main mineral ion found in the sap (7300 µM), followed by calcium (2600 µM), phosphate (2500 µM), nitrate (2300 µM), sulfate (1600 µM), magnesium (500 µM), and chloride (150 µM). The major organic compounds were amino acids (5500 µM) and amides (mainly glutamine, 4000 µM), organic acids (3500 µM, mainly malate), and soluble carbohydrates (500 µM) including glucose, fructose, and sucrose. Over a one-week time course, water flux increased from the first four hours of exudation until 52 hours and then diminished steadily. Mineral ion concentrations remained fairly constant from the onset of the exudation to the end of the collection period, except for potassium which increased continuously and nitrate whose concentration reached a plateau after 24 hours. The high xylem levels of soluble organic compounds (amino acids and soluble carbohydrates), that were found initially, dropped rapidly and then increased reaching a maximum at 52 hours. Finally, the concentration decreased until the end of the exudation period. Furthermore, attention was paid to the effect on the xylem sap composition of recutting the spurs. The data obtained revealed that water flux and soluble carbohydrate release were enhanced, as a result of the recut, up to the maximum level previously reached at 52 hours.
Soluble carbohydrates, particularly oligosaccharides, can take pan in defense responses preventing and restricting fungal pathogen invasion. Morphological changes were observed and ß-glucosidase activity was studied in yellow lupin embryo-axes infected and uninfected with F.oxysporum and cultured for 24 to 96 hours under conditions of a varying carbohydrate supply. The first disease symtoms - necrotic pathological changes were observed 48 hours after the inoculation. A higher intensity of the disease changes as well as growth restriction were found in 72- and 96-h embryo axes growing under carbohydrate deficiency. The activity of â-glucosidase increased during 24 - 96 hours after inoculation. An especially high increase of the activity was noted in 72- and 96-h embryo axes cultured under carbohydrate deficiency. We suggest that sugars are involved in the mechanisms of yellow lupin embryo axis tissues resistance, since they constitute the source of precursors for the synthesis of antimicrobial factors of plant defense response.
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES ABSTRACT The chlorophyll fluorescence measurements were used as a method of assessing the influence of Tetranychus urticae feeding on photosynthetic apparatus of 'Lobo' and 'Jester' apple cultivars. The photochemical efficiency measured with such parameters like: the initial fluorescence (F 0), the maximum fluorescence (F m), the ratio of variable to maximum fluorescence (F v /F m), and the area under the fluorescence curve (S c) decreases continuously with increasing infestation by mites. The only parameter not influenced by mites feeding was the half-rise time from F 0 to F m (T fm). Antennas of both 'Lobo' and 'Jester' cvs. were similarly injured by mites (comparable decrease of F 0) but photochemical reactions of the harvested energy of 'Jester' leaves were less influenced by this pest (lower decrease for F v /F m). It suggests a higher tolerance of the photosynthetic apparatus of 'Jester' cv. to T. urticae feeding, as compared to 'Lobo' cv.
A newly developed fluorescence measuring system is employed for the recording of chlorophyll fluorescence induction kinetics (Kautsky-effect) and for the continuous determination of the photochemical and non-photochemical components of fluorescence quenching. The measuring system, which is based on a pulse modulation principle, selectively monitors the fluorescence yield of a weak measuring beam and is not affected even by extremely high intensities of actinic light. By repetitive application of short light pulses of saturating intensity, the fluorescence yield at complete suppression of photochemical quenching is repetitively recorded, allowing the determination of continuous plots of photochemical quenching and non-photochemical quenching. Such plots are compared with the time courses of variable fluorescence at different intensities of actinic illumination. The differences between the observed kinetics are discussed. It is shown that the modulation fluorometer, in combination with the application of saturating light pulses, provides essential information beyond that obtained with conventional chlorophyll fluorometers.
Chlorophyll (Chi) a fluorescence originates in close vicinity to the sites where light energy is transformed into chemically fixed energy. The
same excitation states that give rise to fluorescence emission also participate in photochemical energy conversion. These
features render Chi fluorescence aunique indicator of photosynthesis. During the past 15 years there has been remarkable progress
in Chi fluorescence research. In practical applications, Pulse-Amplitude-Modulation (PAM) fluorometry in conjunction with
the saturation pulse method has been particularly successful. This chapter outlines the principles of PAM fluorometry and
saturation pulse method. Some examples of typical applications are given. Also the limits of the method are outlined, with
emphasis on the fact that absolute assessment of photosynthetic parameters is complicated by various factors, while relative changes can be assessed with high reliability. Particular attention is given to the theoretical foundation of the method,
at a level, which can be also understood by non-specialists and students. A pivotal role in the determination of Photosystem
(PS) II quantum yield by fluorescence measurements is played by the maximal fluorescence yield, Fm, proper measurement of
which has been a matter of controversy. This is related to a large intrinsic heterogeneity of variable Chi fluorescence, which
is revealed in the polyphasic rise of fluorescence yield upon the onset of saturating light. Arguments are put forward in
favor of Fm determination after full reduction of the plastoquinone pool, including the secondary quinone acceptor of PS II,
QB, the oxidized form of which can support in my view a particular type of nonphotochemical quenching, when the primary quinone
acceptor, QA, is reduced. Possible mechanisms and consequences of this ‘QB-quenching’ are discussed. Some PAM fiuorometers for special applications are described, including systems for phytoplankton
analysis, for investigations at the single cell level (epifluorescence microscopy and microfiber technique) and for imaging
of photosynthetic activity.
Recent developments have altered our view of molecular mechanisms that determine sink strength, defined here as the capacity of non-photosynthetic structures to compete for import of photoassimilates. We review new findings from diverse systems, including stems, seeds, flowers, and fruits. An important advance has been the identification of new transporters and facilitators with major roles in the accumulation and equilibration of sugars at a cellular level. Exactly where each exerts its effect varies among systems. Sugarcane and sweet sorghum stems, for example, both accumulate high levels of sucrose, but may do so via different paths. The distinction is central to strategies for targeted manipulation of sink strength using transporter genes, and shows the importance of system-specific analyses. Another major advance has been the identification of deep hypoxia as a feature of normal grain development. This means that molecular drivers of sink strength in endosperm operate in very low oxygen levels, and under metabolic conditions quite different than previously assumed. Successful enhancement of sink strength has nonetheless been achieved in grains by up-regulating genes for starch biosynthesis. Additionally, our understanding of sink strength is enhanced by awareness of the dual roles played by invertases (INVs), not only in sucrose metabolism, but also in production of the hexose sugar signals that regulate cell cycle and cell division programs. These contributions of INV to cell expansion and division prove to be vital for establishment of young sinks ranging from flowers to fruit. Since INV genes are themselves sugar-responsive “feast genes,” they can mediate a feed-forward enhancement of sink strength when assimilates are abundant. Greater overall productivity and yield have thus been attained in key instances, indicating that even broader enhancements may be achievable as we discover the detailed molecular mechanisms that drive sink strength in diverse systems.
We demonstrate the feasibility of assaying and predicting post-harvest damage in lemons by monitoring chlorophyll (Chl) fluorescence.
Fruit quality was assayed using a commercial instrument that determines photosynthetic performance by imaging Chl fluorescence
parameters under different irradiances. Images of Chl fluorescence from individual lemons reveal that photosynthesis is active
throughout the post-harvest ripening process. Because photosynthesis is highly sensitive to biotic and abiotic stress, variations
in Chl fluorescence parameters over the surface of a lemon fruit can be used to predict areas that will eventually exhibit
visible damage. The technique is able to distinguish between mould-infected areas that eventually spread over the surface
of the fruit, and damaged areas that do not increase in size during ripening. This study demonstrates the potential for using
rapid imaging of Chl fluorescence in post-harvest fruit to develop an automated device that can identify and remove poor quality
fruit long before visible damage appears.
Citrus limon
–mould–
Penicillium digitatum
Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a serious threat to oilseed production in Australia. Eight isolates of S. sclerotiorum were collected from Mount Barker and Walkway regions of Western Australia in 2004. Comparisons of colony characteristics
on potato dextrose agar (PDA) as well as pathogenicity studies of these isolates were conducted on selected genotypes of Brassica napus and B. juncea. Three darkly-pigmented isolates (WW-1, WW-2 and WW-4) were identified and this is the first report of the occurrence of
such isolates in Australia. There was, however, no correlation between pigmentation or colony diameter on PDA with the pathogenicity
of different isolates of this pathogen as measured by diameter of cotyledon lesion on the host genotypes. Significant differences
were observed between different isolates (P ≤ 0.001) in two separate experiments in relation to pathogenicity. Differences were also observed between the different Brassica genotypes (P ≤ 0.001) in their responses to different isolates of S. sclerotiorum and there was also a significant host × pathogen interaction (P ≤ 0.001) in both experiments. Responses between the two experiments were significantly correlated in relation to diameter
of cotyledon lesions caused by selected isolates (r = 0.79; P < 0.001, n = 48). Responses of some genotypes (e.g., cv. Charlton) were relatively consistent irrespective of the isolates of the pathogen
tested, whereas highly variable responses were observed in some other genotypes (e.g., Zhongyou-ang No. 4, Purler) against
the same isolates. Results indicate that, ideally, more than one S. sclerotiorum isolate should be included in any screening programme to identify host resistance. Unique genotypes which show relatively
consistent resistant reactions (e.g., cv. Charlton) across different isolates are the best for commercial exploitation of
this resistance in oilseed Brassica breeding programmes.
Some leading characteristics and historical notes on Botrytis spp. are described here. Botrytis spp. infect many host plants in all climate areas of the world, infecting mainly upper plant parts at preand post-harvest
stages. Bulbs, seeds and other propagation material also suffer infection. Infection can occur in high humidity in the presence
or absence of water films. Infection may be quiescent, aggressive, restricted or widely developing. The production of high
numbers of conidia poses a long lasting threat to susceptible hosts. Genotypic and phenotypic variation is most important
in the broad spectrum pathogen B. cinerea. Moreover, changes in populations in response to selection by exposure to xenobiotics, especially fungicides, are quite common
in the genus and fungicide resistance has been recorded in Botrytis populations throughout the history of the modern fungicide era. Detailed studies on the precise conditions that promote infection,
disease development and survival of inoculum have provided the essential epidemiological information required for design of
control strategies. For example, cultural methods have been developed that increase aeration and drying of the plant canopy
to reduce the risk of Botrytis epidemics. The increasing requirement for alternative approaches to reduce farmers' dependency on use of fungicides led to
the evaluation and exploitation of potential biocontrol agents capable of substantial disease suppression in a commercial
context, and within integrated crop management systems.
Infection processes were examined to investigate the breach by a strain of Leptosphaeria maculans of anatomical barriers in cv. Surpass 400, a cultivar containing single dominant gene-based resistance (SDGBR). Two strains,
UWA 192 and UWA P11, were used to inoculate cvs. Surpass 400 and Westar. The pre-penetration and penetration behaviour of
both strains was similar in both cultivars. However, they differed significantly after penetration. When UWA P11 infected
cv. Surpass 400 through stomata, guard cells rapidly died within a few hours and the surrounding mesophyll cells became necrotic,
constituting a hypersensitive reaction (HR). Hyphal growth continued, albeit slowly, through the intercellular palisade mesophyll
and spongy mesophyll spaces, but hyphae rarely spread beyond the HR region, and did not sporulate. Polyphenolic compounds
accumulated in the area bordering the HR. However, when UWA 192 infected through stomata, symptoms were not evident until
10–12days postinoculation (dpi) and were typically characterized by pale tan to grey circular lesions in which abundant pycnidia
were produced by 14dpi. Subsequently, hyphae extensively spread beyond the lesion border, reaching the veins and progressing
down the petiole towards the stem. Where the SDGBR remained effective (i.e. against strain UWA P11) death of cells was restricted
to a few palisade cells within the HR, even though hyphae were present in the lower tissue layers of the cotyledon. In contrast,
where the SDGBR was not present (cv. Westar) or was overcome (cv. Surpass 400 with UWA 192), extensive death of epidermal
and upper and lower palisade cells occurred throughout infected areas of the cotyledon, with subsequent abundant production
of pycnidia. Polyphenolic compounds, which are also associated with resistance, did not accumulate in this instance. It was
evident that the ability of the host to instigate the HR mechanism displayed by cv. Surpass 400 was lost with UWA 192 resulting
in a “normal” susceptible response. This is the first study of the specific processes involved in the breaching of the HR
in cv. Surpass 400.
Sucrose and monosaccharide transporters mediate long distance transport of sugar from source to sink organs and constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Even if numerous families of plant sugar transporters are defined; efflux capacities, subcellular localization and association to membrane rafts have only been recently reported. On the fungal side, the investigation of sugar transport mechanisms in mutualistic and pathogenic interactions is now emerging. Here, we review the essential role of sugar transporters for distribution of carbohydrates inside plant cells, as well as for plant-fungal interaction functioning. Altogether these data highlight the need for a better comprehension of the mechanisms underlying sugar exchanges between fungi and their host plants.
Botrytis cinerea is a major crop pathogen infesting >220 hosts worldwide. A cryptic species has been identified in some French populations but the new species, B. pseudocinerea, has not been fully delimited and established. The aim of this study was to distinguish between the two species, using phylogenetic, biological, morphological, and ecological criteria. Multiple gene genealogies confirmed that the two species belonged to different, well-supported phylogenetic clades. None of the morphological criteria tested (spore size, germination rate, or mycelial growth) was able to discriminate between these two species. Sexual crosses between individuals from the same species and different species were carried out. Only crosses between individuals from the same species were successful. Moreover, population genetics analysis revealed a high level of diversity within each species and a lack of gene flow between them. Finally, a population survey over time showed that B. cinerea was the predominant species but that B. pseudocinerea was more abundant in spring, on floral debris. This observation could not be explained by temperature adaptation in tests carried out in vitro or by aggressiveness on tomato or bean leaves. This study clearly establishes that B. cinerea and B. pseudocinerea constitute a complex of two cryptic species living in sympatry on several hosts, including grapevine and blackberry. We propose several biological or molecular tools for unambiguous differentiation between the two species. B. pseudocinerea probably makes a negligible contribution to gray mold epidemics on grapevine. This new species has been deposited in the MycoBank international database.
Background:
Herbivory reduces leaf area, disrupts the function of leaves, and ultimately alters yield and productivity. Herbivore damage to foliage typically is assessed in the field by measuring the amount of leaf tissue removed and disrupted. This approach assumes the remaining tissues are unaltered, and plant photosynthesis and water balance function normally. However, recent application of thermal and fluorescent imaging technologies revealed that alterations to photosynthesis and transpiration propagate into remaining undamaged leaf tissue.
Scope and conclusions:
This review briefly examines the indirect effects of herbivory on photosynthesis, measured by gas exchange or chlorophyll fluorescence, and identifies four mechanisms contributing to the indirect suppression of photosynthesis in remaining leaf tissues: severed vasculature, altered sink demand, defence-induced autotoxicity, and defence-induced down-regulation of photosynthesis. We review the chlorophyll fluorescence and thermal imaging techniques used to gather layers of spatial data and discuss methods for compiling these layers to achieve greater insight into mechanisms contributing to the indirect suppression of photosynthesis. We also elaborate on a few herbivore-induced gene-regulating mechanisms which modulate photosynthesis and discuss the difficult nature of measuring spatial heterogeneity when combining fluorescence imaging and gas exchange technology. Although few studies have characterized herbivore-induced indirect effects on photosynthesis at the leaf level, an emerging literature suggests that the loss of photosynthetic capacity following herbivory may be greater than direct loss of photosynthetic tissues. Depending on the damage guild, ignoring the indirect suppression of photosynthesis by arthropods and other organisms may lead to an underestimate of their physiological and ecological impacts.
Plant sucrose transporter activities were shown to respond to changes in the extracellular pH and redox status, and oxidizing compounds like glutathione (GSSG) or H(2)O(2) were reported to effect the subcellular targeting of these proteins. We hypothesized that changes in both parameters might be used to modulate the activities of competing sucrose transporters at a plant/pathogen interface. We, therefore, compared the effects of redox-active compounds and of extracellular pH on the sucrose transporters UmSRT1 and ZmSUT1 known to compete for extracellular sucrose in the Ustilago maydis (corn smut)/Zea mays (maize) pathosystem.
We present functional analyses of the U. maydis sucrose transporter UmSRT1 and of the plant sucrose transporters ZmSUT1 and StSUT1 in Saccharomyces cerevisiae or in Xenopus laevis oocytes in the presence of different extracellular pH-values and redox systems, and study the possible effects of these treatments on the subcellular targeting. We observed an inverse regulation of host and pathogen sucrose transporters by changes in the apoplastic pH. Under none of the conditions analyzed, we could confirm the reported effects of redox-active compounds.
Our data suggest that changes in the extracellular pH but not of the extracellular redox status might be used to oppositely adjust the transport activities of plant and fungal sucrose transporters at the host/pathogen interface.
The role of photosynthesis in plant defense is a fundamental question awaiting further molecular and physiological elucidation. To this end we investigated host responses to infection with the bacterial pathogen Xanthomonas axonopodis pv. citri, the pathogen responsible for citrus canker. This pathogen encodes a plant-like natriuretic peptide (XacPNP) that is expressed specifically during the infection process and prevents deterioration of the physiological condition of the infected tissue. Proteomic assays of citrus leaves infected with a XacPNP deletion mutant (DeltaXacPNP) resulted in a major reduction in photosynthetic proteins such as Rubisco, Rubisco activase and ATP synthase as a compared with infection with wild type bacteria. In contrast, infiltration of citrus leaves with recombinant XacPNP caused an increase in these host proteins and a concomitant increase in photosynthetic efficiency as measured by chlorophyll fluorescence assays. Reversion of the reduction in photosynthetic efficiency in citrus leaves infected with DeltaXacPNP was achieved by the application of XacPNP or Citrus sinensis PNP lending support to a case of molecular mimicry. Finally, given that DeltaXacPNP infection is less successful than infection with the wild type, it appears that reducing photosynthesis is an effective plant defense mechanism against biotrophic pathogens.
In 1931, using their eyes as instruments, H . Kautsky and A . Hirsch related the time course of chlorophyll a fluorescence with photosynthesis in a less-than-one-page article in Naturwissenschaften (see Kautsky's photograph). Chlorophyll a fluorescence is now being used by hundreds of investigators as a probe for various aspects of photosynthesis-from excitation energy transfer in picosecond time scale to CO2 fixation in minutes . It is not only a much used, but also a much abused, tool. It is used because of it being a non-invasive, rapid and a highly sensitive probe, and misused because it is sometimes not recognised that it is affected by various photosynthetic and other reactions. I submit that, like any other technique, if it is used with care and with due regard for its time dependence and competing parameters. it will remain as the one-most powerful tool for probing excitation energy transfer, primary photochemistry, electron flow on both the donor and the acceptor side of photosystem II (PSII) of oxygenic PSII. Further, it is very useful in the quick assay of PSII mutations, and down- regulation and other adjustments to stress (excess light, heat, heavy metal, nutrients and certain herbicides). In this paper, I will present my viewpoint, not a review, on the conceptual and experimental developments in this field. Whenever appropriate, and without any shame and humility, I will include some of my involvement in the excitement surrounding this field. I hope that this paper will serve as a starting point for further discussion of not only the history, but the practical use of chlorophyll a fluorescence as an intrinsic probe of stresses to plants, as well as individual reactions of oxygenic photosynthesis, when combined with other parallel measurements.
Stripe rust is the most serious wheat disease worldwide. Puccinia striiformis f. sp. tritici (Pst), which is the causal agent of stripe rust, is an obligate biotrophic fungus and must take up nutrients from the host through its haustoria. Recent studies have indicated that sugar is the main nutrient taken up by pathogens from host cells. However, the response of sugars to Pst infection in wheat is not well known. In this study, the levels of water-soluble carbohydrates in Pst-infected wheat leaves were investigated, and the results revealed slight changes for all of the carbohydrates except sucrose. Furthermore, the sucrose levels increased in both the compatible and incompatible systems. The photosynthetic rates of infected leaves were determined and found to be significantly increased during the early stage of Pst infection. In addition, the expression profiles of genes associated with photosynthesis were assayed through real-time PCR, and the trends found were identical to that observed for the photosynthetic rate in Pst-infected wheat leaves. These findings indicate that Pst infection can upregulate genes involved in photosynthesis to promote the photosynthetic rate and ultimately induce sucrose accumulation in infected wheat leaves.
A series of experiments is presented investigating short term and long term changes of the nature of the response of rate of CO2 assimilation to intercellular p(CO2). The relationships between CO2 assimilation rate and biochemical components of leaf photosynthesis, such as ribulose-bisphosphate (RuP2) carboxylase-oxygenase activity and electron transport capacity are examined and related to current theory of CO2 assimilation in leaves of C3 species. It was found that the response of the rate of CO2 assimilation to irradiance, partial pressure of O2, p(O2), and temperature was different at low and high intercellular p(CO2), suggesting that CO2 assimilation rate is governed by different processes at low and high intercellular p(CO2). In longer term changes in CO2 assimilation rate, induced by different growth conditions, the initial slope of the response of CO2 assimilation rate to intercellular p(CO2) could be correlated to in vitro measurements of RuP2 carboxylase activity. Also, CO2 assimilation rate at high p(CO2) could be correlated to in vitro measurements of electron transport rate. These results are consistent with the hypothesis that CO2 assimilation rate is limited by the RuP2 saturated rate of the RuP2 carboxylase-oxygenase at low intercellular p(CO2) and by the rate allowed by RuP2 regeneration capacity at high intercellular p(CO2).
: Infection with phloem limited Abutilon Mosaic Virus caused localized carbohydrate accumulation (high levels of starch, sucrose, and hexoses) in leaves of Abutilon striatum during early symptom development. In mature leaves with attenuated symptoms, tissues showing faint vein-clearing had markedly higher carbohydrate contents than uniformly green areas of the same leaf. A similar pattern of carbohydrate accumulation was found in pale-green mosaics in mature leaves with overt symptoms when compared to green-islands of the same leaf, but overnight carbohydrate loses were comparable to controls. Because leaves with attenuated symptoms showed no further symptom development whereas the pale-green mosaics became yellow and eventually necrotic in leaves with overt symptoms, it seems unlikely that carbohydrate accumulation following impaired translocation was responsible for symptom expression. High carbohydrate status in leaves with attenuated symptoms had little effect on nonphotochemical quenching during early stages of photosynthetic induction. In leaves with overt symptoms, areas of high carbohydrate status with pale-green mosaics showed markedly slower nonphotochemical quenching. Early symptom areas of young leaves, and advanced symptom areas of mature leaves had low starch contents but were otherwise similar to controls in carbohydrate status. Impaired nonphotochemical quenching in these tissues tended to reflect the state of symptom development, rather than carbohydrate status. Plants with overt symptoms grew about half as fast as plants with attenuated symptoms.
Kabatiella caulivora is a serious pathogen of clover (Trifolium) spp. Subterranean clover (T. subterraneum) cv. Woogenellup was inoculated with K. caulivora, to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.
Infection of Senecio squalidus by Albugo tragopogonis and of Tussilago farfara and Poa pratensis by Puccinia poarum results in an increase in the activity of an acid invertase localized at the sites of infection. It is concluded that sucrose from the host is first hydrolysed and then absorbed by the parasite. Invertase also plays a key role in the provision of substrate for the typical accumulation of starch around pustules of biotrophic fungi on host species which store this polysaccharide. Compartmentation of invertase and the enzymes concerned with synthesis of sucrose and fructans permits these carbohydrates to be accumulated within pustules on host species which normally store fructans.
Resistance to fungicides is an evolutionary process resulting from the selection of advantageous genotypes in naturally diverse populations. Seven fungicide modes of action are authorised to control grey mould caused by Botrytis cinerea on grapevine in France, and five of them have encountered specific resistance, with variable frequencies in populations and possible consequences for field fungicide efficacy. Moreover, multidrug resistance is caused by fungicide efflux and allows a weak resistance towards six unrelated modes of action. Here, a review is given of the fungicide resistance status of B. cinerea in France, particularly in the vineyards of Champagne, which are the most affected. Recently developed resistance and recent findings concerning the associated resistance mechanisms are focused upon in particular. Finally, antiresistance strategies are presented, and examples of managed resistance are discussed in a more general manner with the aim of extending this knowledge to other crops and countries undergoing similar resistance problems. © 2013 Society of Chemical Industry.
Phytopathogen infection leads to changes in secondary metabolism based on the induction of defence programmes as well as to changes in primary metabolism which affect growth and development of the plant. Therefore, pathogen attack causes crop yield losses even in interactions which do not end up with disease or death of the plant. While the regulation of defence responses has been intensively studied for decades, less is known about the effects of pathogen infection on primary metabolism. Recently, interest in this research area has been growing, and aspects of photosynthesis, assimilate partitioning, and source-sink regulation in different types of plant-pathogen interactions have been investigated. Similarly, phytopathological studies take into consideration the physiological status of the infected tissues to elucidate the fine-tuned infection mechanisms. The aim of this review is to give a summary of recent advances in the mutual interrelation between primary metabolism and pathogen infection, as well as to indicate current developments in non-invasive techniques and important strategies of combining modern molecular and physiological techniques with phytopathology for future investigations.
Invertase (β-fructofuranosidase, EC 3.2.1.26) activity was shown to be stimulated in grape berries after infection with Botrytis cinerea. By using organism-specific extraction methods, evidence was found proving that both partners contribute to the increase in activity. Qualitative analysis of the extracts by SDS–PAGE showed a new invertase species in the botrytised material, with a molecular weight similar to that of Botrytis invertase (BIT). A method allowing the preparative isolation of homogeneous invertase from liquid cultures of B. cinerea in only three steps (ethanol precipitation, DEAE anion exchange and hydroxyapatite chromatography) with 50% recovery was developed. BIT appeared to be strongly glycosylated; interestingly, the amount of glycan seemed to vary and had a remarkable influence on the chromatographic behaviour of the enzyme. After chemical deglycosylation with TFMS, the BIT peptide was used for the production of polyclonal antibodies in chickens (anti-BIT-IgY). The antibodies recognised glycosylated as well as deglycosylated BIT, a partial denaturation of the protein being necessary for reaction. This indicates that the deglycosylation procedure had been successful and that the antibodies were in fact directed to the peptide moiety. However, after a short incubation at 70°C, native BIT was detectable by anti-BIT-IgY, while still active. Western blotting with extracts of diseased berries confirmed the fungal origin of the new invertase form. The anti-BIT-IgY proved highly specific, although some cross-reaction with a protein in Monilia laxa extracts occurred. The importance of careful immunogen preparation in the production of specific antibodies, and the potential of BIT as a target molecule for the immunological detection of B. cinerea are discussed.
The infection of plants with pathogens results in the induction of defence reactions as well as changes in carbohydrate metabolism. On the one hand, the pathogen attempts to manipulate the carbohydrate metabolism of the plant for its own advantage. On the other, the plant has to reorganize carbon fluxes to ensure fight against the pathogen. In order to further investigate the connection between pathogen infection and carbohydrate metabolism, the effects of two types of pathogen, biotrophic and necrotrophic, on gene expression, endogenous sugar levels and photosynthesis of tomato plants were analysed. Photosynthetic gene expression was downregulated on infection with Pseudomonas syringae and Botrytis cinerea. In contrast, expression of a sink-specific gene encoding a cell wall invertase and of defence genes was induced by both pathogens. These results provide evidence for a co-regulation of defence, sink and photosynthetic gene expression in planta in response to both types of pathogen. The brassinosteroid-containing plant restorative ComCat enhanced resistance against B. cinerea and counter-regulated the repression of photosynthetic gene expression. Endogenous sugar levels decreased and the hexose to sucrose ratio increased on treatment with B. cinerea. The application of chlorophyll fluorescence imaging revealed the spatio-temporal heterogeneity of the pathogen response. At 24 h after infection, inhibition of photosynthetic electron transport was restricted to the direct vicinity of the infection site, which was surrounded by a circle of increased photosynthetic activity. The photosynthesis of the remaining leaf was not affected at this stage. These results show the usefulness of chlorophyll fluorescence imaging for the assessment of the complex spatio-temporal changes and for the definition of the areas relevant for other types of determination, e.g. gene expression.
The book is the result of intensive work of 43 authors, all of them leading scientists in the Botrytis sciences. Each chapter describes a particular aspect of fungal biology and its impact on disease processes and host response. New technologies have arisen that when applied to long-standing problems or to test new hypotheses have been most rewarding and many of these are covered in this book. The chapters are cross linked so that readers can follow associated material. The twenty inter-connected chapters of the book are grouped according to three major themes: the fungus and its pathogenicity factors; plant reactions to infection; and epidemiology and management of important Botrytis-incited diseases. This book adopts a multidisciplinary approach to integrate the state-of-the-art knowledge in all key areas of common interest in the fungi and their plant interactions. The book includes detailed reviews of Botrytis spp. and the diseases they cause in plant systems. It also provides a comprehensive description of these fungal necrotrophs, including their diversity of response to the environment, their speciation and relatedness, sources of variation for evolution and mole.
- The fungal metabolite botrydial was detected for the first time in ripe fruits of sweet pepper (Capsicum annuum) wound-inoculated with conidial suspensions of Botrytis cinerea and also in leaves of Phaseolus vulgaris and Arabidopsis thaliana inoculated without wounding. This phytotoxin was produced in soft rot regions of the infection. In C. annuum, the most aggressive isolate produced the highest botrydial concentrations in planta. The levels of botrydial produced by this isolate did not correlate with the reported relative susceptibilities of four P. vulgaris genotypes. The results suggest that botrydial is a pathogenicity factor for this fungus, but not a primary determinant of pathogenicity.
The metabolic and cellular changes in source leaves of Nicotiana tabacum L. cv SNN during an incompatible interaction with Phytophthora nicotianae van Breda de Haan were investigated and compared with defence reactions. Hypersensitive cell death was preceded by a rapid and highly localized shift to non-assimilatoric metabolism. During the first 6 h post infection (hpi), reactive oxygen species (ROS) accumulated. Callose was deposited at the interface of adjacent mesophyll cells (≥1 hpi), the export of sucrose collapsed and its content in the apoplast increased. Stomata closed and photosynthetic flux was reallocated from CO2 assimilation in favour of photorespiration. This was accompanied by an increase in respiration, glucose-6-phosphate dehydrogenase (G6PDH) activity, apoplastic invertase and hexose content. Later (>6 hpi) the photosynthetic electron transport chain was interrupted and photosynthesis completely collapsed. This was accompanied by a further increase in apoplastic invertase and carbohydrates, respiration and oxidative pentose phosphate pathway (OPPP) and followed by further burst in ROS release. Hypersensitive cell death did not appear until photosynthesis completely declined. Photosynthesis was visualized by chlorophyll-a fluorescence imaging on a macro- and microscopic scale. Decline in photosynthesis and defence reactions were highly localized processes, which occur in single mesophyll cells. We propose that in photoautotrophic leaves, photosynthesis and assimilatory metabolism must be switched off to initiate respiration and other processes required for defence. An early blockage of intercellular sugar transportation, due to callose deposition, in conjunction with enhanced apoplastic invertase activity could facilitate this metabolic shift.
Kinetic fluorescence imaging was used to set a new detection limit for plant exposure to low levels of destruxins – phytotoxins of Alternaria brassicae. A general experimental algorithm is presented that can be used to identify the combination of fluorescence parameters providing the highest contrast between the affected and unaffected plants or plant segments. Leaves of canola (Brassica napus) and white mustard (Sinapis alba) were exposed to various concentrations of destruxins and images of key fluorescence signals (F0, FM, FP, and of FS) were captured in a single kinetic experiment. Contrast was quantified within these images between the leaf areas exposed to destruxins and the untreated areas. The highest contrast was found in the image constructed by pixel-to-pixel division of images F0 by FP and F0 by FM. Using the F0/FM ratio image, we were able to detect exposure to destruxin concentration as low as approximately 0.05 mg l−1 applied to canola leaf and approximately 10 mg l−1 when applied to mustard. The detection limits were significantly lower than those obtained by optical microscopy indicating that kinetic chlorophyll fluorescence imaging can be used as a diagnostic tool in screening for varieties with an enhanced resistance to destruxins of Alternaria brassicae.
Albugo candida (Pers.) (O.) Kunze is a biotrophic pathogen which infects the crucifer Arabidopsis thaliana (L.) Heynh forming discrete areas of infection. Eight days after inoculation of leaves, white blisters became visible on the under surface of the leaf although no symptoms were apparent on the upper surface. By day 14, the region of leaf invaded by fungal mycelium had become chlorotic. Recently it has been hypothesized that an accumulation of soluble carbohydrates, following an increase in invertase activity, may trigger sugar signal transduction pathways leading to the repression of photosynthetic gene expression and to the induction of defence proteins. This hypothesis was investigated by quantifying localized changes in carbohydrate and photosynthetic metabolism and the expression of genes encoding photosynthetic and defence proteins. Quantitative imaging of chlorophyll fluorescence revealed that the rate of photosynthesis declined progressively in the invaded regions of the leaf. However, in uninfected regions of the infected leaf the rate of photosynthesis was similar to that measured in the control leaf until late on during the infection cycle when it declined. Images of nonphotochemical fluorescence quenching (NPQ) suggested that the capacity of the Calvin cycle had been reduced in infected regions and that there was a complex metabolic heterogeneity within the infected leaf. A. candida also caused localized changes in the carbohydrate metabolism of the leaf; soluble carbohydrates accumulated in the infected region whereas the amount of starch declined. The reverse was seen in uninfected regions of the infected leaf; carbohydrates did not accumulate until late on during infection and the amount of starch increased as the infection progressed. There was an increase in the activity of invertases which was confined to regions of the leaf invaded by the fungal mycelium. The increase in apoplastic invertase activity was of host origin, as mRNA levels of the ATβFRUCT1 gene (measured by semiquantitative RT-PCR) increased 40-fold in the infected region. The increase in soluble invertase activity resulted from the appearance of a new isoform in the invaded region of the leaf. Current evidence suggests that this was of fungal origin. Northern blot analysis of cab and rbcS showed that photosynthetic gene expression was repressed in the infected leaf from 6 days after inoculation (DAI) when compared to control leaves. In contrast, there was no detectable induction of defence proteins in the infected leaf. These data are discussed in the context of the sugar-sensing hypothesis presented above.
Infection of bean leaves by Colletotrichum lundemuthianum causes vein necrosis and subsequent localized wilting of the blade. The effect of infection on photosynthesis was investigated by imaging leaf chlorophyll fluorescence as a means of mapping stomatal and metabolic inhibition of photosynthesis. During infection, CO2 assimilation (An), stomatal conductance to water vapour, and photosynthetic electron transport rate (Jt) decreased, whereas dark respiration increased. An decreased more than was expected from the reduction in green leaf area, showing that photosynthesis was inhibited in apparently healthy areas. Under subsaturating irradiance, images of Jt in air showed that photosynthesis decreased gradually, with this effect shifting from green to necrotic areas. Sudden increase in CO2 concentration to 0·74% in the atmosphere around the leaf only partially reversed this inhibition, showing that both stomatal and metabolic inhibition occurred. Under limiting irradiance, decreases in Jt and in maximal Jt during high CO2 exposure as leaf damage severity increased suggested that metabolic inhibition was mediated through an inhibition of Ribulose 1·5-bisphosphate (RuBP) regeneration. Finally, the importance of our data in terms of assessing the loss of photosynthetic yield from visible symptoms – as is currently performed in epidemiology – is discussed.
The consequences of biotic stress have been poorly understood, partly because its application is difficult to control and partly because its physiological consequences are highly variable. Many plant viruses are recognised on the basis of leaf symptoms that depend on localised changes to chloroplast structure and function. This paper reviews recent progress in understanding early interactions between plant viruses and the photosynthetic apparatus, using chlorophyll fluorescence analysis of novel, defined algal-virus systems and using high resolution imaging of chlorophyll fluorescence and other photosynthetic processes in higher plant systems. We then consider the consequences of viral effects on photosynthetic functioning for whole plants and populations with an emphasis on the potential interactions with other environmental factors. Early responses indicated by increase in both non-photochemical quenching of fluorescence and increased reduction state of the primary electron transport acceptor QA suggest that, not surprisingly, both photoprotective and photoinhibitory processes contribute to the accelerated local demise of the photosynthetic apparatus and symptom development. In other cases, localised accumulations of carbohydrate and source-sink imbalance following infection may inhibit gene expression, leading to altered levels of chloroplast protein complexes and enzymes of photosynthetic metabolism coincident with symptom development. Recent experiments suggest that much of the variability in plant responses to biotic stress may result from interactions with other environmental factors, such as light intensity and nutrition. Experiments suggest that virus infections may have greater effects on fitness and competitive ability in low N, high light environments than in shaded, high nutrient conditions. Some ecological implications of these observations are discussed.
ContentsSummaryI. IntroductionII. Important tools for exprimentation with Ustilago myadisIII. Cell fusion requres a complex signalling networkIV. Development of the dikaryon: the bE/bW complex at workV. A connection between cell cycle, morphogenesis and virulenceVI. The early infection stagesVII. Proliferation and differentiaton in the plant hostVIII. The Ustilago maydis genomeIX. Conclusions
AcknowledgementsReferencesSummaryThe smut fungus Ustilago maydis is a ubiquitous pathogen of corn. Although of minor economical importance, U. maydis has become the most attractive model among the plant pathogenic basidiomycetes under study. This fungus undergoes a number of morphological transitions throughout its life-cycle, the most prominent being the dimorphic switch from budding to filamentous growth that is prerequisite for entry into the biotrophic phase. The morphological transition is controlled by the tetrapolar mating system. Understanding the mating system has allowed connections to signalling cascades operating during pathogenic development. Here, we will review the status and recent insights into understanding pathogenic development of U. maydis and emphasize areas and directions of future research.
Nine microsatellite markers were characterized in the fungus Botrytis cinerea. Genomic DNA sequences from the partial sequencing of 12 000 bacterial artificial chromosome (BAC) clones, were screened by BLAST for various microsatellite motives, and primer pairs were designed. Cross-amplification and polymorphism were assessed on 49 isolates from B. cinerea and two related species, collected from natural populations on several plants and locations.
The infection of host plants by Botrytis spp. is mediated by numerous extracellular enzymes and metabolites. Each of these compounds may play a role in different
stages of the infection process. Cutinases, lipases and some cell wall-degrading enzymes may facilitate the penetration of
the host surface, while toxins, oxalate and reactive oxygen species may contribute to killing of the host cells. Several cell
wall-degrading enzymes contribute to the conversion of host tissue into fungal biomass, but also other enzymes, such as laccases
and proteases are potentially involved in pathogenesis. The cloning of the corresponding genes in recent years has facilitated
studies on gene expression and targeted mutagenesis. This chapter gives an updated overview of the research performed on these
secreted enzymes and metabolites and the role they play in pathogenesis.
Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.
Recent progress in chlorophyll fluorescence research is reviewed, with emphasis on separation of photochemical and non-photochemical quenching coefficients (qP and qN) by the saturation pulse method. This is part of an introductory talk at the Wageningen Meeting on The use of chlorophyll fluorescence and other non-invasive techniques in plant stress physiology. The sequence of events is investigated which leads to down-regulation of PS II quantum yield in vivo, expressed in formation of qN. The role of O2-dependent electron flow for pH- and qN-formation is emphasized. Previous conclusions on the rate of pseudocyclic transport are re-evaluated in view of high ascorbate peroxidase activity observed in intact chloroplasts. It is proposed that the combined Mehler-Peroxidase reaction is responsible for most of the qN developed when CO2-assimilation is limited. Dithiothreitol is shown to inhibit part of qN-formation as well as peroxidase-induced electron flow. As to the actual mechanism of non-photochemical quenching, it is demonstrated that quenching is favored by treatments which slow down reactions at the PS II donor side. The same treatments are shown to stimulate charge recombination, as measured via 50 s luminescence. It is suggested that also in vivo internal thylakoid acidification leads to stimulation of charge recombination, although on a more rapid time scale. A unifying model is proposed, incorporating reaction center and antenna quenching, with primary control of pH at the PS II reaction center, involving radical pair spin transition and charge recombination to the triplet state in a first quenching step. In a second step, triplet excitation is trapped by zeaxanthin (if present) which in its triplet excited state causes additional quenching of singlet excited chlorophyll.
Localised changes in photosynthesis in oat leaves infected with the biotrophic rust fungus Puccinia coronata Corda were examined at different stages of disease development by quantitative imaging of chlorophyll fluorescence. Following inoculation of oat leaves with crown rust the rate of whole-leaf gas exchange declined. However, crown rust formed discrete areas of infection which expanded as the disease progressed and these localised regions of infection gave rise to heterogeneous changes in photosynthesis. To quantify these changes, images of chlorophyll fluorescence were taken 5, 8 and 11 d after inoculation and used to calculate images representing two parameters; II, a measure of PSII photochemical efficiency and Fm/Fm, a measure of non-photochemical energy dissipation (qN). Five days after inoculation, disease symptoms appeared as yellow flecks which were correlated with the extent of the fungal mycelium within the leaf. At this stage, II was slightly reduced in the infected regions but, in uninfected regions of the leaf, values of II were similar to those of healthy leaves. In contrast, qN (Fm/Fm) was greatly reduced throughout the infected leaf in comparison to healthy leaves. We suggest that the low value of qN in an infected leaf reflects a high demand for ATP within these leaves. At sporulation, 8 d after inoculation, II was reduced throughout the infected leaf although the reduction was most marked in areas invaded by fungal mycelium. In the infected leaf the pattern of non-photochemical quenching was complex; qN was low within invaded regions, perhaps reflecting high metabolic activity, but was now much higher in uninfected regions of the infected leaf, in comparison to healthy leaves. Eleven days after inoculation green islands formed in regions of the leaf associated with the fungal mycelium. At this stage, photosynthesis was severely inhibited over the entire leaf; however, heterogeneity was still apparent. In the region not invaded by the fungal mycelium, II and qN were very low and these regions of the leaf were highly fluorescent, indicating that the photosynthetic apparatus was severely damaged. In the greenisland tissue, II was low but detectable, indicating that some photosynthetic processes were still occurring. Moreover, qN was high and fluorescence low, indicating that the cells in this region were not dead and were capable of significant quenching of chlorophyll fluorescence.
Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.
The effects of powdery mildew (Erysiphe graminis f. sp. tritici ) on the carbohydrate metabolism of wheat leaves was studied during infection. During infection the triazole fungicide hexaconazole (trade name ANVIL, Zeneca Agrochemicals) was applied to leaves to determine if removal of the fungal sink reversed any pathogen-induced alterations in host physiology. Five days after inoculation there was a large accumulation of sucrose, glucose, fructose and hexose-phosphates and an increased partitioning of current photosynthate into starch rather than sucrose in both mildewed leaves and those treated with fungicide 2 and 3 days after inoculation. Within 48 h of inoculation, acid invertase activity bad doubled and this increase was maintained until 7 days after inoculation. Application of hexaconazole to a mildewed leaf caused a transient decline in invertase activity but over subsequent days activity increased again until it resembled that observed in a mildewed leaf. Hexaconazole severely inhibited fungal development and hence the size of the fungal sink. This suggests that the enhanced invertase activity was triggered by a signal from the fungus but was not directly related to the size of the fungal sink. These results suggest that (i) the stimulation of invertase activity in mildewed leaves altered the source-sink relationship of the leaf leading to an accumulation of soluble carbohydrates and a decrease in sucrose synthesis and (ii) that application of hexaconazole successfully prevented development of the fungus but that the metabolic alterations which occurred following the initial infection were not reversible within the time scale studied.
Proteins are implicated in the foam stabilization of Champagne wines. They may have a grape, yeast, bacteria or fungal origin. Botrytis cinerea is a widespread fungal pathogen, which is the causal agent for gray mold. The first part of this work showed the deleterious effect of the presence of this fungus on the foaming properties of a champenois base wine. Foamability and foam stability were reduced, respectively, by 47.7% and 33.3% in the botrytized wine, as compared to the healthy wine. In a second part, SDS–PAGE and two-dimensional electrophoresis (2-DE), coupled with immunodetection, were used to study (thoroughly) the protein patterns of both wines. With 2-DE and silver-staining detection, the disappearance of numerous spots, located in an acidic pH range, was observed. Indeed, the number of spots detected was about two times more abundant in the healthy wine than in the botrytized one, suggesting that a proteolysis occurred. On the other hand, the presence of new proteins, likely fungal proteins, proteins secreted by the plant as a response to B. cinerea infection, or even protein fragments resulting from partial proteolysis, was detected in the botrytized wine. All these modifications of the wine protein content were undoubtedly due to the presence of B. cinerea and this might be a reason for the loss of foaming properties of Champagne base wines, though no relationship between these two phenomena can be established from the results obtained.
Measurements of the quantum yields of chlorophyll fluorescence and CO2 assimilation for a number of plant species exposed to changing light intensity and atmospheric CO2 concentrations and during induction of photosynthesis are used to examine the relationship between fluorescence quenching parameters and the quantum yield of non-cyclic electron transport. Over a wide range of physiological conditions the quantum yield of non-cyclic electron transport was found to be directly proportional to the product of the photochemical fluorescence quenching (qQ) and the efficiency of excitation capture by open Photosystem II (PS II) reaction centres (Fv/Fm). A simple fluorescence parameter, ΔφF/φFm, which is defined by the difference in fluorescence yield at maximal φFm, and steady-state φFs, divided by φFm, can be used routinely to estimate changes in the quantum yield of non-cyclic electron transport. It is demonstrated that both the concentration of open PS II reaction centres and the efficiency of excitation capture by these centres will determine the quantum yield of non-cyclic electron transport in vivo and that deactivation of excitation within PS II complexes by non-photochemical processes must influence the quantum yield of non-cyclic electron transport.
In a compatible interaction biotrophic fungi often lower the yield of their hosts by reducing photosynthesis and altering the fluxes of carbon within the infected leaf. In contrast, comparatively little is known about the metabolic consequences of activating resistance responses. In this study we investigated the hypothesis that the activation of both race-specific ( Mla12 ) and broad-spectrum ( mlo ) resistance pathways in barley leaves infected with Blume- ria graminis represents a cost to the plant in terms of carbon production and utilization. We have shown, using quantitative imaging of chlorophyll fluorescence, that dur- ing a susceptible interaction, photosynthesis was progres- sively reduced both in cells directly below fungal colonies and in adjacent cells when compared with uninoculated leaves. The lower rate of photosynthesis was associated with an increase in invertase activity, an accumulation of hexoses and a down-regulation of photosynthetic gene expression. During both Mla12 - and mlo -mediated resis- tance, photosynthesis was also reduced, most severely inhibited in cells directly associated with attempted pene- tration of the fungus but also in surrounding cells. These cells displayed intense autofluorescence under ultraviolet illumination indicative of the accumulation of phenolic compounds and/or callose deposition. The depression in photosynthesis was not due only to cell death but also to an alteration in source-sink relations and carbon utiliza- tion. Apoplastic (cell wall-bound) invertase activity increased more rapidly and to a much greater extent than in infected susceptible leaves and was accompanied by an accumulation of hexoses that was localized to areas of the leaf actively exhibiting resistance responses. The accumula- tion of hexoses was accompanied by a down-regulation in the expression of Rubisco ( rbcS ) and chlorophyll a/b bind- ing protein ( cab ) genes (although to a lesser extent than in a compatible interaction) and with an up-regulation in the expression of the pathogenesis-related protein 1 ( PR-1 ). These results are consistent with a role for invertase in the