Role of a phytoalexin in controlling lesion development in leaves of Viciafaba after infection by Botrytis spp

Phytoalexin extracted from infection droplets and diseased tissues behaved as an ether-soluble acid, and was easily separated by solvent partition from other ether-soluble substances. The phytoalexin was formed in leaves by apparently healthy cells in advance of hyphae of either Botrytis fabae or B. cinerea, and in response to physical injury. Concentrations of phytoalexin around deep lesions caused by B. fabae were completely fungistatic. B. fabae caused apparent degradation of phytoalexin in lesions, and removed phytoalexin from solutions in vitro much more readily than did B. cinerea. The lower sensitivity to the phytoalexin, and the possibly related greater ability to metabolize the phytoalexin, are major factors in the greater pathogenicity of B. fabae than of B. cinerea. The same properties largely explain the ability of B. fabae to cause the so-called ‘aggressive’ phase of the chocolate-spot disease under some conditions.     

Some Characteristics of Red Light-induced Substance(s) against Botrytis cinerea Produced in Broad Bean Leaflets

The red light-induced antifungal substance(s) produced in broad bean was of relatively high molecular weight, water soluble, heat stable and fungi specific. Cellulose thin layer chromatography (TLC) of infection droplets of Botrytis cinerea or water droplets without spores of B. cinerea, recovered from inoculated broad bean leaflets kept under red light for 48 h, displayed inhibition zones at approximate Rf values of 0.0 and 0.6. Inhibition zones observed in cellulose TLC of water droplets were relatively faint compared to those of infection droplets. In a time-course study of accumulation of the antifungal substance(s), antifungal activity in both water and infection droplets recovered from red light irradiated broad bean leaflets occurred after 24 h irradiation. However, the antifungal activity in infection droplets was significantly higher than in water droplets. The antifungal substance(s) was less active against Botrytis fabae than B. cinerea.     

The rates of fungal development and lesion formation in leaves of Vicia faba during infection by Botrytis cinerea and Botrytis fabae

Loss of the water droplet above inoculation sites during the first day after inoculation inhibited lesion formation by Botrytis cinerea and prevented the development of spreading lesions of B. fabae. With droplets present two general patterns of infection by B. cinerea were determined; in one, few or no symptoms were produced and in the other, limited lesions developed with marked browning of the inoculation site. Where few or no symptoms were produced, germination and germ-tube growth were inhibited on the leaf surface. B. cinerea was inhibited within the leaf at sites bearing limited lesions; invading hyphae were restricted to brown epidermal cells. Fungal growth on the leaf surface was greatest at sites with most browning beneath the droplet area. Variation in lesion development by B. cinerea could not be related to droplet position or leaf damage during normal preparation for inoculation. Plants differed in their susceptibility to lesion formation by B. cinerea. B. fabae, with droplet present, was not inhibited on the leaf surface and spread inter- and intra-cellularly beneath the inoculum drop and then into surrounding tissues. Delay in spread until the inoculation site was completely necrotic and colonized suggested that B. fabae is partially inhibited during the initial phase of infection. The rate of lesion spread varied in different plants and was most rapid in the youngest leaves.     

Changes in wyerone acid concentrations in leaves of Vicia faba after infection by Botrytis cinerea or B.fabae

Wyerone acid was produced by leaves of Vicia faba in response to infection by both Botrytis cinerea and B.fabae. Host cell death caused by either fungus appeared to be the trigger for rapid wyerone acid synthesis, although the phytoalexin was not confined to brown cells. At B. cinerea inoculation sites wyerone acid concentration increased rapidly, at the time of fungal invasion of the epidermis, to levels greater than that completely inhibitory to mycelial growth. Wyerone acid is therefore probably the primary cause of the inhibition of B. cinerea within infected tissue. The partial blackening of B.fabae inoculation sites and surrounding peripheral tissues was accompanied by an increase in wyerone acid. There followed a striking decrease as tissues became completely blackened and invaded by B.fabae. B.fabae appeared to metabolize wyerone acid and prevent its accumulation in invaded tissues. Mycelial growth of B. fabae was less sensitive to wyerone acid than was B. cinerea. The differing abilities of B.fabae and B. cinerea to spread from lesions after both have induced wyerone acid production probably depend on both their differing sensitivities to the phytoalexin and their abilities to metabolize it to less toxic products.     

Splash dispersal of fungus spores and fungicides in the laboratory and greenhouse

A laboratory technique is described for the production of drops of simulated rain in which fungal spores were suspended. When such drops containing conidia of Botrytis fabae impacted on a target leaf the secondary droplets produced infections on receptor broad bean leaves. The capacity of fungicides applied to the target leaf to redistribute in secondary splash droplets was examined in terms of the infectivity of the spores in the droplets. The extent to which a copper fungicide reduced infection on the receptor leaves was related to the level and tenacity of the fungicide deposit on the target leaf. The effect of wetting agents on the redistribution of this fungicide could probably be explained by their influence on the tenacity of the initial deposit. In general the capacity of different fungicides to inhibit infection by the secondary droplets was related to the inherent toxicity of the fungicides to B. fabae. Implications of the dispersal of spores and fungicides by rain splash are briefly considered with reference to field conditions.     

Characterization of antifungal glycoprotein in red-light-irradiated broadbean leaflets

 Red-light treatment of broadbean leaflets resulted in the production of antifungal substance(s) against Botrytis cinerea. The antifungal substance(s) was positively charged, as the antifungal constituent was removed by the cation exchanger CM cellulose. Treatment of infection droplets with glycosidases (α-mannosidase, β-galactosidase, β-glucosidase), glycol-specific reagent periodate (NaIO₄), and proteinase K completely eliminated antifungal activity, suggesting that both protein and carbohydrate are active components. The protein content of infection droplets was 0.148 mg/ml. The HPLC gel column analysis of infection droplets resulted in four fractions; all the fractions showed antifungal activity. Received: June 14, 2002 / Accepted: August 12, 2002 Correspondence to:Y. Honda     

Identification of Botrytis spp. on Plants Grown in Iran

A total of 363 isolates were collected from all over Iran. They were isolated from apple, arum lily, briar rose, bride wort, broad bean, camellia, canola, carnation, cucumber, egg plant, feijoa, geranium, gerbera, gladiolus, grape, guilder rose, hibiscus, iris, kiwifruit, oleander, onion, orange, pear, pomegranate, primrose, quince, redbud, robinia, rose, rubber plant, sow thiste, spathe flower, strawberry, tomato, violet, wall flower and wheat. To identify the species, morphological characters such as conidiophore length, conidial and sclerotial dimensions were measured. According to morphological and cultural characters, eight Botrytis species were identified: B. aclada sensu lato, B. cinerea, B. fabae, B. convoluta, B. gladiolorum, B. paeoniae, B. pelargonii and B. porri. As far as we are aware, this is the first report of the last five species from Iran. These species were examined by polymerase chain reaction (PCR) using necrosis and ethylene‐inducing protein (NEP2) and C729 primers. A 835 bp band was amplified in B. cinerea, B. fabae and B. pelargonii, using NEP2, but not in others. However, C729 primers amplified a 700 bp band in B. cinerea and B. pelargonii and a 600 bp in B. fabae.     

Effects of Botrytis fabae infection and mechanical defoliation on seed yield of field beans (Vicia faba)

Effects on seed yield of mechanical defoliation and inoculation with Botrytis fabae were compared using pot-grown plants of Vicia faba (cv. Maris Beagle). Treatments, which were made at the end of flowering, were applied singly and in all combinations to leaves (a) below, (b) at, and (c) above the flowering nodes (i.e. 2³ factorial). Yield was unaffected by treatments applied to leaves below the flowering nodes. Removal of leaves at flowering nodes did not reduce the number of pods but yield was reduced because there were fewer and smaller seeds. Inoculation of this zone also reduced yield; pods were lost at some nodes but it could also be shown that, irrespective of pod loss, yield at individual nodes was reduced in proportion to the severity of infection on leaves at the same nodes. Removal of leaves above flowering nodes reduced yield almost to the same extent as removal of leaves at flowering nodes but inoculation resulted in only a small amount of infection and yield was not reduced significantly. These results, taken in conjunction with recent studies on the physiology of the host plant, show that beans are exceptionally vulnerable to attack by B. fabae at the stage of flowering and early pod development. At later stages of development infection is unlikely to have a substantial effect on yield.     

Proteomic analysis of the phytopathogenic fungus Botrytis cinerea during cellulose degradation

The ascomycete Botrytis cinerea is a phytopathogenic fungus infecting and causing significant yield losses in a number of crops. Moreover, in the last few years, B. cinerea has been adopted as an important model system in molecular phytopathology. In spite of these contributions, the molecular basis of the infection cycle remains unclear. Proteomic approaches have revealed significant information about the infective cycle of several pathogens, including B. cinerea. The main aim of this study is to make available a proteomic database containing a significant number of identified proteins from B. cinerea. In brief, three independent B. cinerea cultures supplemented with carboxymethylcellulose were used, and the extracted proteins were independently separated by 2‐D PAGE to obtain the proteome map from B. cinerea. Two hundred and sixty‐seven spots were selected for MALDI TOF/TOF MS analysis, resulting in 303 positive identifications, mostly representing unannotated proteins. Identified proteins were then classified into categories using the PANTHER classification system ( www.pantherdb.org ), showing the relevance of protein metabolism and modification process and oxidoreductase activity. Since cellulose is one of the major components of the plant cell wall, many of the identified proteins may have a crucial role in the pathogenicity process. In brief, this proteomic map of B. cinerea will be a useful basis for exploring the proteins involved in the infection cycle, which will in turn provide new targets for crop diagnosis and focused fungicide design.     

Application of benzothiadiazole and Trichoderma harzianum to control faba bean chocolate spot disease and their effect on some physiological and biochemical traits

Chocolate spot disease is the most prevalent and important disease in the major faba bean growing regions in the world. Different concentrations of the abiotic inducer (0.3 and 0.5 mM benzothiadiazole) and the biotic inducer (1 × 10⁷ and 2 × 10⁷ spore/ml Trichoderma harzianum) were used alone or in combination to study their efficiency against faba bean chocolate spot disease caused by Botrytis fabae and Botrytis cinerea and their effect on some chemical analyses (phenylalanine ammonia lyase activity, total flavonoids and peroxidase isozymes, pectin and lignin content and total chlorophyll content). Application of the tested inducers as foliar treatment significantly reduced the severity of chocolate spot disease as compared with untreated infected plants. The reduction in disease severity was associated with a gradual increase in phenylalanine ammonia lyase activity. Maximum increase was recorded at 72 h after inoculation with B. fabae and B. cinerea. In addition, the levels of flavonoids in induced infected leaves recorded a sharp increase at 24 h after inoculation with B. fabae or B. cinerea. Also, pectin and lignin contents in the cell wall of induced infected plants were significantly increased as compared with untreated infected plants. Beside the induction of resistance, the tested inducers markedly increased total chlorophyll content in treated infected plants as compared with untreated infected plants. Isozymes analysis revealed that new peroxidase bands were induced only in treated faba bean leaves in response to infection with B. fabae or B. cinerea.     

Presence of Transposons and Mycoviruses in Botrytis cinerea Isolates Collected from a German Grapevine Growing Region

Transposons and infection of fungal strains with mycoviruses can have significant effects on distinctive phenotypic traits of phytopathogenic fungi such as mycelial growth and sporulation, pathogenicity or fungicide resistance. Two transposable elements (TE), Boty and Flipper, are known to be associated with the ubiquitous fungus Botrytis cinerea. In addition, the presence of two types of ssRNAsRNA viruses, BVX and BVF, has been reported in B. cinerea. In this study, we assessed the genetic diversity of B. cinerea isolates, all sampled within a small‐sized German viticultural area (‘Rheingau’) by examining and classifying them according to the presence of TEs and mycoviruses. A subset of the isolates was further analysed with microsatellite markers to determine the origin of particular isolates with or without one or both mycoviruses. Virtually all isolates (98%) sampled in two different years (2008 and 2010) were screened positive for the presence of a transposon. Presence of one or both B. cinerea mycoviruses was confirmed for 37% of the analysed isolates sampled in 2010, representing the first record of B. cinerea mycoviruses in German isolates. Assignment on individual B. cinerea isolates to different genetic groups was independent of the presence or absence of a mycovirus or a transposable element, respectively. Furthermore, we found no correlation between the presence of either a mycovirus or a transposable element and different viticultural management practices, soil properties or levels of nitrogen fertilization applied to the respective vineyards. However, mycelial growth of B. cinerea strains containing mycovirus BVF was significantly reduced at lower temperatures.     

Infection of Arabidopsis with a necrotrophic pathogen,Botrytis cinerea,elicits various defense responses but does not induce systemic acquired resistance (SAR)

Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species. In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells. Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms. We analyzed the role of SAR in B. cinerea infection of Arabidopsis. We show that although B. cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B. cinerea infections. Induction of SAR with avirulent P. syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B. cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B. cinerea disease symptoms. In addition, we show that B. cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways. Thus, B. cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.     

The Influence of o-hydroxyethylorutin on Botrytis cinerea Oxidant–Antioxidant Activity

The objective of this study was to evaluate the effects of o‐hydroxyethylorutin on Botrytis cinerea mycelium growth and metabolism. Hydrogen peroxide concentration, superoxide dismutase, catalase and peroxidase activities were compared in the pathogens’ mycelium grown on control and o‐hydroxyethylorutin containing medium. Transfer of B. cinerea mycelium to medium supplemented with 5 mm o‐hydroxyethylorutin resulted in a large decrease in catalase activity. No changes in mycelium growth, hydrogen peroxide concentration and superoxide dismutase activity were observed. Guaiacol and ascorbate peroxidases were not detected in mycelia. The data are consistent with previous findings that o‐hydroxyethylorutin treatment of tomato plants restricts the development of B. cinerea infection due to the induction of higher active oxygen species (AOS) generation in plants by this compound. Being poor in catalase, the pathogen may not be able to cope with increasing AOS formation. The results indicate that catalase is an infective agent of B. cinerea.     

Conversion of wyerone to wyerol by Botrytis cinerea and B. fabae in vitro

The phytoalexin wyerone was induced to accumulate in cotyledons of Vicia faba infected with Botrytis cinerea or B. fabae. The acetylenic keto ester, wyerone, was converted to the less antifungal corresponding hydroxy ester, wyerol, by both species of Botrytis in vitro.     

A proteomic study of pectin‐degrading enzymes secreted by Botrytis cinerea grown in liquid culture

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty‐seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.     

Dihydrowyerone derivatives as components of the furanoacetylenic phytoalexin response of tissues of Vicia faba

Changes in concentrations of 7 wyerone derivatives in bean tissues undergoing resistant reactions to Botrytis cinerea or B. fabae and in cotyledons in response to mercuric chloride have been examined using high performance liquid chromatography. The proportion of derivatives occurring in their saturated (dihydro) forms varied between cotyledon, leaf and pod tissues and with time after inoculation. Unsaturated derivatives were always present in greater concentrations than their dihydro analogues.     

Effect of membrane integrity on survival competition of Botrytis cinerea upon QoI fungicide pyraclostrobin

Botrytis cinerea, the causal agent of the grey mould disease, developed resistance to multiple fungicides. However, the role of cell membrane in survival competition of B. cinerea upon quinone outside inhibitor (QoI) fungicide has not yet been elucidated. In this paper, the enhancement of cystamine, a transglutaminase inhibitor, on membrane integrity of B. cinerea was determined, and the effect of the enhancement on the sensitivity of B. cinerea to pyraclostrobin was investigated. The results showed that pyraclostrobin inhibited mycelial growth with EC₅₀ as 1.122 and 3.042 μg/ml at 24 and 48 hr, respectively. In the treatment of 5 and 50 μg/ml pyraclostrobin, membrane integrity of B. cinerea was broken, causing high permeability, lipid peroxidation, flocculent and malformed surface with vague septum and abundant agglomerates inside and outside the mycelia. Cystamine even at 50 and 200 μg/ml had little inhibitory effect on mycelial growth. However, in presence of 50 or 200 μg/ml cystamine, the mycelia from pyraclostrobin treatment possessed a significantly reduced leakage, lower MDA content, and a revived fibrous and transparent surface. Meanwhile, SEM images showed that membrane integrity of the mycelia was significantly improved and the agglomerates were dramatically disappeared. Synergy assays further revealed that B. cinerea regained less sensitivity to pyraclostrobin inhibition. In conclusion, membrane integrity controls mycelia sensitivity and is required for survival competition of B. cinerea upon pyraclostrobin.     

Wyerone Acid Phytoalexin Synthesis and Peroxidase Activity as Markers for Resistance of Broad Beans to Chocolate Spot Disease

Infection of broad bean (Vicia faba L.) leaves with Botrytis fabae (Sard.) resulted in wyerone acid phytoalexin accumulation and increase in peroxidase activities in resistant and susceptible broad bean cultivars. Rapid wyerone acid accumulation was observed in leaves of resistant cultivars that reached levels greater than twofold of the susceptible cultivars. Following infection of broad bean cultivars, either resistant or susceptible to B. fabae, wyerone acid synthesis in resistant cultivars peaked at 5 days then declined, whereas in susceptible cultivars synthesis gradually decreased. While the ethanol‐extract of wyerone acid significantly reduced B. fabae spore germination with increased concentrations, the phytoalexin had no effect on in vitro B. fabae germ‐tube growth. Peroxidase activity in infected leaves of resistant was 10 times higher than the susceptible cultivars and eight times higher in uninfected leaves of the resistant than the susceptible cultivars. Peroxidase activity in infected leaves was greater than twofold in resistant cultivars and less than twofold in susceptible cultivars, when compared with uninfected leaves. The ratio of peroxidase activity in infected to uninfected leaves increased over time in susceptible cultivars but remained unchanged in resistant cultivars. Peroxidase activity in uninfected and infected leaves and wyerone acid biosynthesis in infected broad bean plants were successfully used to ascertain the resistance and susceptibility of four broad bean cultivars to B. fabae. Using wyerone acid synthesis and peroxidase activity as preliminary markers for resistance of broad bean to chocolate spot disease, caused by B. fabae, is suggested in this study.