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Many fungi adhere strongly to an outer surface of a host or a substratum before penetration; the ability to adhere is a virulence factor for plant and animal pathogens. Because substrata such as the plant cuticle are largely inert hydrophobic surfaces, plant pathogenic fungi often adhere to plastics. Strong adhesion is generally mediated by a secreted glue. Fungal glues are typically formed in an aqueous environment, and as such they may provide models for commercial glues, including for biomedical applications. The best-characterized fungal glue is a galactosaminogalactan polymer that serves as an adhesive to plastic, fibronectin, and epithelial cells in the mammalian pathogen Aspergillus fumigatus. However, most fungal glues are apparently mannoprotein(s). Despite our advances in fungal genomics, most fungal glues are uncharacterized, in contrast to the well-characterized fungal pathogen-mammalian host adhesins produced by Candida albicans; these adhesins, which bind to specific host receptors, are primarily large, glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins that are posttranslationally covalently cross-linked to the cell wall β1,6-glucans. Adhesins typically have tandem repeats; adhesins such as the C. albicans ALS family bind to both specific cell types and to plastic. We postulate that the mannoprotein glues from plant pathogenic fungi are secreted in a more water-soluble form, are transiently sticky, and are cross-linked extracellularly. Anti-adhesive strategies may enable new plant disease control methods that are environmentally compatible because anti-adhesive strategies do not necessarily require any uptake of compounds into the fungus or disruption of a eukaryotic metabolic pathway.

Sorghum ( Sorghum bicolor L.) produces 3-deoxyanthocyanidin phytoalexins. They accumulate as inclusions in cells and inhibit infection in a site-specific response. Inclusions are self-organizing 3-deoxyanthocyanidins. In aqueous solution, protons and acetate counter-ions condition self-stacking. Non-vesicular cytoplasmic trafficking of inclusions to sites of ingress is discussed. It is suggested that re-arrangements in plant cells under attack direct accumulation of inclusions to penetration sites. Nuclear migration, cytoplasmic streaming and intracellular pH provide an environment for inclusion trafficking and release of the phytoalexins. Luteolinidin, when self-organized as pigmented inclusions, mediates disruption of plant and fungal plasma membranes as well as reconstructed bilayer liposomes.

In sorghum, ingress of Cochliobolus heterostrophus stimulates the synthesis of 3-deoxyanthocyanidins that act as phytoalexins. Apigeninidin and luteolinidin are two major phytoalexins induced in the first 24 h after infection. In an attempt to understand genetic regulation of the biosynthesis of sorghum phytoalexins, we isolated a differentially expressed partial cDNA. Characterization and comparison showed that this cDNA sequence corresponds to a putative flavonoid 3′-hydroxylase . Full length sequence characterization allowed us to establish that the sorghum putative f3′h cDNA encodes a peptide of 517 amino acids that has domains conserved among cytochrome P450 proteins functioning in the flavonoid biosynthetic pathway. Heterologous expression of the putative f3′h cDNA in Escherichia coli yielded a membrane preparation that catalyzed the hydroxylation of naringenin. We show here that transcription of the flavonoid 3′-hydroxylase was coordinately regulated with that of chalcone synthase and dihydroflavonol reductase, and expression of these genes was induced within the first 24 h of fungal challenge. Synthesis of apigeninidin and luteolinidin followed the induced expression of the f3′h gene, implicating its role in fungal induced expression of sorghum phytolaexins.

Conidia of Cochliobolus carbonum secrete a toxin (HC-toxin) during appressorium formation on maize leaves. Plasma desorption mass spectrometry revealed that approximately 70 ng of toxin per 106 conidia were secreted during the first 16 h of morphogenesis. Growth of the fungus was monitored microscopically. Extensive fungal growth occurred in the susceptible interaction by 24 h. In spite of a substantial amount of HC-toxin, the fungus failed to become established in the resistant host even after 36 h. Results suggest that the resistance conditioned by Hm1, which encodes a toxin reductase, causes inactivation of the toxin early in the interaction.

The concentration of 3-deoxyanthocyanidins from inoculated sorghum exhibited a sigmoid curve for accumulation between 10 and 32 h after inoculation. Luteolinidin and apigeninidin were the most abundant deoxyanthocyanidins present. Methoxy-apigeninidin and the caffeic acid ester of arabinosyl 5-O-apigeninidin were detected but their concentrations were very low. At the time of inoculation, conidia of Cochliobolus heterostrophus were brown and cells were turgid. Host tissue was colorless and no symptoms were evident. Spectrophotometry and high performance liquid chromatography (HPLC) analyses showed that the 3-deoxyanthocyanidin phytoalexins were not present at the time of inoculation. Spectrophotometry did not detect deoxyanthocyanidins in plant extracts until 10–12 h post inoculation. In contrast, HPLC analysis showed that inoculated tissue released small amounts of luteolinidin and apigeninidin by 4 h after inoculation. It is the speed of this response that is unusual. After inoculation, both host cells and conidia appeared red. Phytoalexin-containing inclusions were released from the host tissue and became associated with the extracellular sheath of the dead conidia.

Recent use of Sorghum bicolor as a target for grass genomics has presented new resources for gene discovery in novel metabolic pathways in Poaceae. Sorghum synthesizes a unique class of flavonoid phytoalexins, the 3-deoxyanthocyanidins, in response to fungal infection. The biosynthetic pathways for 3-deoxyflavonoids are largely uncharacterized but are known to involve transcriptional activation of chalcone synthase (CHS). CHS, or naringenin CHS, catalyses the formation of naringenin, the precursor for different flavonoids. We have isolated seven sorghum CHS genes, CHS1–7, from a genomic library on high-density filters. CHS1–7 are highly conserved and closely related to the maize C2 and Whp genes. Several of them are also linked in the genome. These findings suggest that they are the result of recent gene duplication events. Expression of the individual CHS genes was studied in silico by examination of expressed sequence tag (EST) data available in the public domain. Our analyses suggested that CHS1–7 were not differentially expressed in the various growth and developmental conditions represented by the cDNA libraries used to generate the EST data. However, we identified a CHS-like gene, CHS8, with significantly higher EST abundance in the pathogen-induced library. CHS8 shows only 81–82% identity to CHS1–7 and forms a distinct subgroup in our phylogenetic analysis. In addition, the active site region contains substitutions that distinguish CHS8 from naringenin CHS. We propose that CHS8 has evolved new enzymatic functions that are involved in the synthesis of defence-related flavonoids, such as the 3-deoxyanthocyanidins, during fungal infection.

Wright, A. J., Thomas, B. J., Kunoh, H., Nicholson, R. L., Carver, T. L. W. (2002). Influences of substrata and interface geometry on the release of extracellular material by Blumeria graminis conidia. Physiological and Molecular Plant Pathology, 61, (3), 163-178. Sponsorship: Aventis Crop Science / MAFF / BBSRC

The 3-deoxyanthocyanidin phytoalexins produced in sorghum leaves in response to Colletotrichum sublineolum have chemical structure similarities to the 3-deoxy flavonoids that are precursors of phlobaphene pigments. Phlobaphenes are commonly observed in the pericarp of mature sorghum grains, while synthesis of 3-deoxyanthocyanidin phytoalexins is a site-specific response to infection with C. sublineolum. We have taken a genetic approach to investigate the possible overlap between the two sub-branches of flavonoid biosynthesis in sorghum that lead to phlobaphenes and 3-deoxyanthocyanidin phytoalexins. A sorghum line with a functional y1 gene synthesizes 3-deoxyanthocyanidins as well as phlobaphenes. However, a progenitor line with the mutable Y1-candystripe (Y1-cs) allele shows variable levels of biosynthesis of these compounds. The Y1-cs allele carries a copy of the Candystripe1 (Cs1) transposable element in the y1 gene. We demonstrate here that the variability in the expression of 3-deoxyanthocyanidins produced in individual mesocotyls of hyper-mutable Y1-cs plants is a function of the activity of the y1 gene. TheCs1 insertion in the Y1-cs allele blocks y1 function, while excision of Cs1 out of they1 locus restores the gene to a functional state. The combined molecular and biochemical characterization of sibling plants confirms that the allelic state of the y1 gene is completely correlated with the production of phytoalexins in response to fungal infection. These results provide support for the idea that the y1 gene regulates the biosynthesis of both 3-deoxyanthocyanidin phytoalexins and phlobaphene pigments in sorghum.

The extracellular matrix (ECM) released by conidia of Pestalotia malicola when incubated on artificial and natural substrata was investigated. Scanning electron microscopy (SEM) images indicated the presence of ECM beneath conidial appendages. Stains for non-specific esterase activity revealed that enzyme activity was initially present at the conidial appendages. Gelatin peel tests observed with light microscopy and SEM indicated that a strong initial spore adhesion occurs at the appendages flanking the conidium body. That the ECM was seen beneath firmly adhered conidial appendages suggests that it has a role in adhesion. Harvested ECM material from germinating conidia was deglycosylated and acetylated for GC/MS monosaccharide identification. Mole percent calculations show mannose to be the predominant monosaccharide present.

The release of extracellular matrix (ECM) and the emergence of germ tubes from conidia ofBlumeria graminis were studied by light microscopy and micromanipulation. More prompt and frequent ECM release was confirmed on an artificial hydrophobic substratum than on an artificial hydrophilic substratum. Conidia initially incubated on the hydrophilic substratum were transferred by micromanipulation to either the hydrophobic or the hydrophilic substrata. Immediately after transfer onto the hydrophobic substratum, 75% of conidia released ECM, whereas only 16% did so upon transfer to the hydrophilic substratum. Conidia transferred onto the hydrophobic substratum produced a primary germ tube (PGT) more promptly and frequently than those transferred to the hydrophilic substratum. Thus, conidia recognize and respond to substratum hydrophobicity perhaps immediately after contact. When inoculated onto either isolated barley cuticle or the hydrophobic artificial substratum, 2/3 of the conidia produced a PGT from their polar regions. By contrast, on the hydrophilic substratum 2/3 of conidia did so from the side region. These results show that substratum hydrophobicity affects the location of PGT emergence from conidia. Furthermore, the study indicates that very rapid recognition of surface hydrophobicity by conidia promotes ECM release and this in turn may influence the location of PGT emergence.

The development of appressoria by germinating Blumeria graminis conidia depends on its germ tubes making contact with the host surface. Low angle, low temperature scanning electron microscopy showed that 80% or more of first-formed germ tubes of f. spp. hordei, tritici and avenae conidia emerged from close to the host leaf surface, and so made contact with it allowing them to become functional primary germ tubes. Light microscopy of f. sp.hordei confirmed this result and, in addition, showed that germ tubes frequently emerged close to, and contacted, various hydrophobic and hydrophilic artificial substrata. Geometric models of conidium-substratum interfaces were developed and a “surface point method” was derived to predict the frequency with which contact would result if germ tube emergence was a random phenomenon. However, observed contact frequencies were far higher (c. three to eight times) than predicted. Thus, the germ tube emergence site was determined as a response to substratum contact. In part, this appeared to be a non-specific response. Nevertheless, germ tube contact frequencies were greater on the curved surface of leaf epidermal cells than on planar surface, suggesting that specific recognition of leaf surface characteristics may promote directional emergence. The area of contact required to stimulate directional germ tube emergence was very small: contact with a microneedle tip or with a spiders’ suspension thread was sufficient for many conidia. Similarly, on leaves, the only contact is between the tips of a limited number of conidial wall projections and the edges of epicuticular leaf wax plates. Micromanipulation to roll conidia so that their original site of contact with a leaf was rotated away from it, led to the majority of first-formed germ tubes growing away from the leaf, i.e. emerging close to the site of original contact. The experiments indicated that the site of germ tube emergence is determined within 1 min of deposition. This implicated the release of conidial extracellular materials in recognition of the conidium-leaf surface contact site.

Sorghum (Sorghum bicolor) synthesizes a complex mixture of 3-deoxyanthocyanidin phytoalexins in response to inoculation with the non-pathogenic fungus Bipolaris maydis. The anthocyanin cyanidin 3-dimalonyl glucoside, is also synthesized naturally in response to light. To determine the order and time of appearance of these compounds, etiolated sorghum mesocotyls were inoculated with B. maydis and tissue extracts were analysed by photodiode array-HPLC every 2 h for the first 24 h and at 48 h post inoculation (hpi). Uninoculated and inoculated etiolated mesocotyls were also allowed to incorporate L-[U-14C] phenylalanine. Apigeninidin appeared at 10 hpi, followed by luteolinidin and apigeninidin 5-O-arabinoside at 14 hpi. Luteolinidin 5-methylether was not detected until 18 hpi and apigeninidin 7-methylether not until 20 hpi. The concentrations of the primary phytoalexins, apigeninidin, luteolinidin and apigeninidin 5-O-arabinoside, rose steadily between 12 and 24 hpi, and the levels of apigeninidin and luteolinidin were approximately equivalent by 24 hpi. However, between 24 and 48 hpi luteolinidin and luteolinidin 5-methylether accumulated rapidly so that by 48 hpi the amounts of luteolinidin and luteolinidin 5-methylether had increased approximately twofold. Radiolabelling also showed that 14C was incorporated into the 3-deoxyanthocyanidins and cyanidin 3-dimalonyl glucoside. Several other unidentified phenolic compounds also accumulated radioactivity.

Conidia of the plant pathogen Blumeria graminis recognize and respond to features of certain substrata. Appropriate surfaces induce release of an extracellular proteinaceous matrix from the body of the conidium. Contact with a hydrophobic substratum elicits almost immediate release of the matrix at the contact interface. In this investigation we present, for the first time, evidence that recognition of the substratum by conidia can stimulate uptake of anionic, low-molecular-weight materials before germination. This facilitated transport could be a mechanism for recognition of the host and determination of the direction of growth of the emerging germ tube toward the host leaf surface.

3-Deoxyanthocyanidins are the principal phytoalexins that accumulate in sorghum in response to fungal infection. Two cultivars which differed in their reaction to Colletotrichum sublineolum , the pathogen causing the anthracnose disease in sorghum, were examined for their phytoalexin response and expression of defense-related genes. In the incompatible interaction, fungal development in the host was contained during early stages of pathogenesis. When compared to the susceptible cultivar, defense responses in the resistant cultivar were characterized by a greater and faster accumulation of phytoalexins and an earlier induction of defense-related genes encoding chalcone synthase and pathogenesis-related protein PR-10. In the compatible interaction, the pathogen was able to colonize the host with the proliferation of primary and secondary hyphae. In addition to the quantitative and timing differences, there were qualitative differences between the cultivars in their phytoalexin response. Thus, the resistant cultivar accumulated a complex phytoalexin mixture, including luteolinidin and 5-methoxyluteolinidin, neither of which was produced by the susceptible cultivar following fungal inoculation. These compounds were shown previously to exhibit higher fungitoxicity than other phytoalexin components in sorghum. Our results demonstrate that 3-deoxyanthocyanidin phytoalexins represent major components of resistance to C. sublineolum in sorghum.

The time course of release of an extracellular matrix (ECM) from conidia of the barley powdery mildew pathogen Blumeria graminis was investigated by scanning electron and light microscopy. Conidia released the ECM material preferentially onto a hydrophobic surface and release was detected within 20 s of contact of a conidium with the substratum. No such rapid release of an ECM could be detected when conidia were deposited onto the hydrophilic surface of clean glass. A limited amount of ECM was, however, released from conidia onto the hydrophilic surface of a hydrated cellulose membrane. A time study on hydrophobic plastic revealed that although the ECM was clearly present within the first hour after contact of the conidium with the substratum, it was much reduced in quantity after 12 h incubation. The ECM was demonstrated by micromanipulation and light microscopy to be present as a liquid at the contact interface of the conidium and the substratum. The ECM could not be detected by SEM beneath conidia on leaves, but light microscopy and micromanipulation demonstrated that the ECM could be detected beneath some conidia on the waxy surface of an epidermal strip taken from the abaxial surface of a barley leaf. The ECM material stained positively for protein and could not be removed from the conidium interface by vacuum desiccation in the scanning electron microscope indicating that the ECM is not simply water.

The extracellular matrix (ECM) ofCochliobolus heterostrophus (anamorph:Bipolaris maydis) was made visible by gold/silver and FITC-lectin staining at different stages of germ tube development. A proteinaceous material was released from conidia as germ tubes began to emerge and continued to be released from the germ tube tip throughout elongation. A material that did not stain for protein was observed to surround germ tubes upon their elongation. At later stages of maturation, germ tubes were surrounded by a sheath of proteinaceous material. After 15 h of incubation, staining with the FITC-labeled Concanavalin A revealed that a carbohydrate material surrounded and extended between hyphae. The ECM extract was separated into two fractions which were shown by SDS-PAGE and HPLC analyses to consist of proteins and carbohydrates. The results demonstrate that the composition and physical structure of the ECM change over time. Thus, the ECM is not a static material. Rather, the components of the ECM appear to be laid down at different stages of fungal morphogenesis, possibly related to germ tube emergence, elongation, and maturation.

The mass spectrometric analysis of 3-deoxyanthocyanidins and anthocyanins present in crude extracts from sorghum plant tissue was carried out by matrix-assisted laser desorption ionization mass spectrometry (MALDI). Sensitivities to as low as 5 pmol\ μ l were easily attained for pure samples of the anthocyanidin, pelargonidin, and the anthocyanin, malvin. Sensitivities to the level of 15 pmol\ μ l were attained for 3-deoxyanthocyanidins present in crude extracts from sorghum plant tissue. Through its capability for analyzing very small quantities of these compounds in unpurified samples, MALDI provided a sensitive means for the detection of these flavonoid pigments in plant tissues.

The fungus Pestalotia malicola attacks fruits of Chinese quince ( Chaenomeles sinensis ) in Japan. Scanning electron microscopy (SEM) in combination with micromanipulation as well as cryo SEM demonstrated that the fungus was growing on, within, and beneath the fruit cuticle. The ability of the pathogen to grow in association with the cuticle suggested that the fungus produces cutinase(s) and/or non-specific esterase(s). Enzyme assays revealed that the fungus produced an enzyme, or enzymes, with activities characteristic of a cutinase and a non-specific esterase when cultured on cucumber cutin as the sole carbon source. The same enzyme activities were also demonstrated to be constitutively present in the extracellular matrix that surrounds conidia in conidiomata.

Sorghum (Sorghum bicolor L. Moench) accumulates the anthocyanin cyanidin 3-dimalonyl glucoside in etiolated mesocotyls in response to light. Inoculation with the nonpathogenic fungus Cochliobolus heterostrophus drastically reduced the light-induced accumulation of anthocyanin by repressing the transcription of the anthocyanin biosynthesis genes encoding flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase. In contrast to these repression effects, fungal inoculation resulted in the synthesis of the four known 3-deoxyanthocyanidin phytoalexins and a corresponding activation of genes encoding the key branch-point enzymes in the phenylpropanoid pathway, phenylalanine ammonia-lyase and chalcone synthase. In addition, a gene encoding the pathogenesis-related protein PR-10 was strongly induced in response to inoculation. The accumulation of phytoalexins leveled off by 48 h after inoculation and was accompanied by a more rapid increase in the rate of anthocyanin accumulation. The results suggest that the plant represses less essential metabolic activities such as anthocyanin synthesis as a means of compensating for the immediate biochemical and physiological needs for the defense response.

Cellulase and pectinase activities were examined in the exudates released from conidia of Blumeria graminis (Syn: Erysiphe graminis) and their germlings on two hydrophobic artificial substrata. Conidia were allowed to germinate on the substrata for 1, 6 and 16hr for the formation of primary germ tubes, appressorial germ tubes and maturation of appressoria, respectively. The exudates released by 16hr after inoculation were collected by washing the substrata with 10mM Tris-HCl buffer (pH 7.0). The exudate-buffer mixture was then charged on an anion- (High Q) and a cation- (High S) exchange column. Fractions eluted with a step-wise 0.2-1.0 NaCl gradient from a High Q column exhibited cellulase activity in a carboxylmethyl cellulose plate test and in ρ-nitrophenyl-β-D-cellobioside assay, but not pectinase activity in a polygalacturonic acid-yeast extract-agar plate test. Thus, cellulases are released from germlings by 16hr after incubation on the substrata. The exudates released at 1 and 6hr after inoculation also exhibited a little activity of cellulase but not of pectinase. However, the exudates from the surfaces of conidia on conidiophores which were prepared by washing with 10mM Tris-HCl buffer (pH 7.0) exhibited neither cellulase nor pectinase activities. The exudates prepared by washing conidia and germlings with 1M NaCl contained pectinase but not cellulase activities, suggesting that pectinases may be tightly adsorbed to the surface of the conidium. Homogenates of ungerminated conidia and germlings contained cellulase and pectinase activities, indicating that some of cellulase and pectinases are present constitutively within conidia. This is the first direct evidence that conidia of a powdery mildew fungus produce cellulases and pectinases and that their germlings release cellulases. Since carboxylmethyl cellulose and ρ-nitrophenyl-β-D-cellobioside were used as substrates to detect cellulase activity, the cellulases shown in this paper should be considered to correspond to carboxylmethyl cellulases and exo-β-glucanases.

Aetiolated mesocotyls of sorghum were inoculated with the anthracnose pathogen, Colletotrichum sublineolum, and placed in the light or in the dark for 120 h. A time course study showed that each of the four known 3-deoxyanthocyanidin phytoalexins accumulated regardless of whether seedlings had been incubated in the light or the dark. Analyses of extracts by high performance liquid chromatography and plasma desorption mass spectrometry confirmed the presence of each of the phytoalexins. The phytoalexins were never detected in the control, uninoculated plants, regardless of the light treatment. Total RNA was extracted from the mesocotyls, and a time course of RNA hybridization revealed accumulation of transcripts for phenylalanine ammonia-lyase (PAL) and chalcone synthase at times consistent with those expected to be required for phytoalexin synthesis. PAL transcripts accumulated to higher levels in light-grown than in dark-grown seedlings. Importantly, chalcone synthase transcripts accumulated in response to inoculation and accumulation was independent of light. The accumulation of phytoalexins in the dark and the increase of RNA transcripts question the assumption that light is always required to initiate phenolic compound synthesis at the level of PAL. The results suggest that in sorghum the induction of the synthesis of PAL transcripts and the resultant synthesis of the 3-deoxyanthocyanidin phytoalexins occurs as a response to fungal infection and probably is separate from the induction of PAL and phenolic compound synthesis which occurs as a response to light.

A polyphenol oxidase was demonstrated to be a component of the extracellular conidial mu- cilage of Colletotrichum graminicola. The enzyme was identified as a laccase based on its oxidation of syr- ingaldazine in the absence of hydrogen peroxide and its absorption maximum at 593 nm, that is character- istic of blue, type II copper-containing laccases. The enzyme, purified by DEAE cellulose, Superose and MONO Q Fast Protein Liquid chromatographic pro- cedures, had a molecular weight of 85 kD, a pH op- timum of 6.0, and exhibited K,, and V,,,ax values of 0.214 mmoles and 1.014 mmoles L-' min-~, respec- tively, with syringaldazine as the substrate. The func- tion of the enzyme is discussed relative to its potential role in fungal survival and detoxification of plant phenols.

A method of HPLC analysis of 3-deoxyanthocyanidins was developed which allowed the detection of these phytoalexins by plasma desorption mass spectrometry (PDMS). In addition, the times at which samples were taken were increased to 60h post-inoculation. As a result of the changes in protocol, a new deoxyanthocyanidin was detected. The compound was isolated and purified and found by PDMS to have a molecular weight of 285. In addition to the PDMS data, bathochromic shift analyses indicated that the structure of the compound is consistent with that of a 5-methyl ether of luteolinidin. Fungitoxicity assays demonstrated that the compound is a phytoalexin as it prevented germination by, and killed conidia ofColletotrichum sublineolum, the sorghum anthracnose pathogen, at a concentration of 3μ M . The 5-methoxyluteolinidin exhibited greater fungitoxicity than luteolinidin, a non-methoxylated phytoalexin.

To determine whether the level of phenylalanine ammonia-lyase (PAL) activity in sorghum mesocotyl tissues is elevated in response to inoculation we used sorghum cultivars that differed in their ability to synthesize anthocyanins in response to light. These cultivars provided a means of distinguishing the light induced high background levels of PAL from increases in PAL that occur as a result of attempted infection. The induction of PAL as a response to both light and to attempted fungal infection was further confirmed by a time course study of RNA blot hybridizations with a barley PAL cDNA. Results showed that PAL transcripts in the cultivar that did not synthesize anthocyanins began to accumulate within 3h of exposure of the tissue to light but that the level of the transcript decreased rapidly thereafter. However, when the same cultivar was inoculated with the nonpathogenBipolaris maydisthe intensity of the PAL transcript remained elevated throughout a period of 24h after inoculation. The results demonstrate that in this monocotyledonous host it is necessary to separate the naturally occurring high levels of PAL activity that are induced by light from the induction of PAL enzyme activity which occurs as a response to attempted fungal infection.

The establishment of a fungal pathogen on the surface of its host is essential to the success of the infection process. For many fungi, establishment on the host is an active process that may depend on recognition of the host surface through chemical or topographic signals. Events that allow for establishment may be considered to represent the "preparation of the infection court" by the pathogen. This sometimes involves the adhesion of the pathogen to the host and possibly the alteration of the host's surface topography or chemistry. Adhesion is often presumed to be a single, chemically mediated event associated with germ tube or appressorium formation. However, adhesion of ungerminated propagules may also occur, and evidence suggests that it is mediated by the release of adhesive materials directly from the propagule upon contact with a suitable substratum. Fungi may require either a hydrophobic or a hydrophilic surface to initiate the infection process. The barley powdery mildew pathogen, Erysiphe graminis, requires a hydrophilic surface for appressorium formation, yet the barley leaf is extremely hydrophobic. The problem is resolved by the release of an exudate from conidia that makes the hydrophobic leaf surface hydrophilic. In contrast, Colletotrichum graminicola requires a hydrophobic surface for the initiation of its infection process. Ungerminated conidia of this fungus release materials that allow for the rapid adhesion of conidia, which ensures that germination and appressorium formation occur, initiating the infection process. For both fungi, these events happen well in advance of germination and establish the pathogen at the site of the infection court. Key words: adhesion, cuticle, cutinase, surface hydrophobicity, infection process.

Conidia of Colletotrichum graminicola that had adhered to substrata released materials that were examined by scanning electron microscopy (SEM) or by light microscopy after staining for protein or carbohydrate. SEM of conidia that had adhered to dimethyldichlorosilane-coated or polystyrene-coated glass revealed that material had been released onto the substratum surface where the conidia had been in contact with the surface. Gold/silver staining of adhered conidia revealed patterns of staining that indicated that proteinaceous materials had been released from the tip of the conidium and at the site of contact of the conidium with the substratum (contact interface). When adhered conidia were stained with fluorescein (FITC)-conjugated lectins, only FITC-concanavalin A (FITC-ConA) labelled carbohydrates at the contact interface. FITC-ConA also bound to materials that surrounded conidia that had adhered. Different patterns of labelling by other FITC-conjugated lectins were also observed, indicating that the material released from a conidium has a specific spatial arrangement around the conidium. Both the ConA lectin and the Lens culinaris agglutinin lectin blocked adhesion, indicating that the material released from conidia contained glucose and/or mannose. Together, these results suggest that the material associated with adhered conidia is composed of glycoprotein.

Phenylalanine ammonia-lyase (PAL) activity in barley leaves was measured at intervals after inoculation with the powdery mildew pathogen Erysiphe graminis f.sp. hordei . Measurements were made at times that included the periods of attempted penetration by the E. graminis primary germ tube and appressorium. The results demonstrated that extractable enzyme activity increased at 6 h and between 12-15 h after inoculation, times consistent with attempted penetration by the primary germ tube and the appressorium, respectively. Enzyme activity increased regardless of the resistance or susceptibility of the barley cultivar to the fungus suggesting that the response was non-specific and was not a reflection of the resistance or susceptibility of the cultivar to the pathogen prior to the time of penetration. When barley was inoculated with the nonpathogen E. pisi , only a single period of elevation in PAL enzyme activity was detected. This was consistent with the fact that, unlike E. graminis, E. pisi does not produce a primary germ tube. The enzyme activity increased between 9-15 h after inoculation, consistent with the time of the attempted penetration of the leaf by the E. pisi appressorium. Northern blot analyses to detect the time of appearance of PAL mRNA indicated that the level of the message began to increase at 0·5 h after inoculation with both fungi, and that the intensity of the increase was greatest in response to E. pisi . The results are discussed with respect to the presumed importance of host phenolic compound metabolism that occurs as a response to the fungal infection process.

The influence of age on conidial ultrastructure was investigated in Colletotrichum graminicola. The cytoplasmic volume of young conidia-those harvested from 5-7-d-old cultures-was dominated by lipid bodies. These conidia also were characterized by numerous small vacuoles with electron-dense contents. Other structures present included mitochondria, microbodies, ribosomes, elements of tubular ER, short strands of rough ER, multivesicular bodies, simple cisternal elements, Woronin bodies, microtubules, and large amounts of what appeared to be glycogen. Older conidia harvested from 30-35-d-old cultures exhibited a significantly different ultrastructure. They contained fewer lipid bodies, lacked the large glycogen deposits, were very highly vacuolate, and contained sheetlike strands of ER and extensive aggregations of tubular ER. Structures thought to be autophagic vacuoles also were present. The conidial wall appeared similar in conidia of both ages and consisted of a thicker inner electron-transparent layer ...

Ungerminated conidia of Colletotrichum graminicola , the causal agent of anthracnose leaf blight of corn, adhered to corn leaves within minutes of contact. Adhesion approached 30% of the conidial population by 30 min on the fourth and fifth leaves of 5-week-old plants. In contrast, adhesion of conidia on the eighth or ninth leaf of 8-week-old plants reached a maximum of only 20% by 2 h suggesting that the adhesion of ungerminated conidia to the leaf is influenced by leaf age. Germination of conidia began 6 h after inoculation. Within 3 h after germination was first observed, the percentage adhesion increased and reached a new maximum level, indicating that a second phase of adhesion occurred as a result of germination. These results show that adhesion of ungerminated conidia as well as conidial germlings is essential to the infection process and the successful development of disease by C. graminicola . Scanning electron microscopy and micromanipulation of adhered conidia revealed both the alteration of the leaf surface to which the conidia had been adhered and the presence of a possible adhesive material on the contact surface of the ungerminated conidia and the contact surface of the conidial germ tube.

Conidia of Colletotrichum graminicola , the causal agent of corn anthracnose, begin to adhere to hydrophobic surfaces immediately after contact with the surface and hours before the onset of germination. Conidia rarely adhered to glass, a hydrophilic surface. The maximum percentage adhesion of ungerminated conidia was approx. 30–40% on polystyrene Petri dishes and 20–25% on dimethyldichlorosilane (DMS)-coated glass slides. The maximum adhesion on both surfaces was attained within 30 min of the time of contact. Thus, conidia appear to require hydrophobic surfaces for adhesion. The extracellular mucilage that is produced in association with conidia was shown to not be involved in adhesion. Adhesion was not influenced by conidium concentration but was influenced by conidium age. Exposure of conidia to different temperatures in adhesion assays delayed the ability of conidia to adhere. The respiration inhibitor, sodium azide and the transcription inhibitor, actinomycin D, had no effect on adhesion. In contrast, inhibitors of glycoprotein transport (brefeldin A) and protein synthesis (cycloheximide) reduced conidial adhesion by 30 and 50%, respectively. Treatment of conidia with pronase E prevented adhesion completely. If conidia were pre-treated with pronase E and then washed free of the enzyme, approx. 20 % of the conidial population recovered the ability to adhere. Conidia treated with Concanavalin A at a concentration of 1 mg ml −1 also failed to adhere. This effect was partially reversed by the haptens glucose and mannose. Together, these results suggest that glycoprotein synthesis is involved in the adhesion of ungerminated conidia.

A technique for the mass spectrometric analysis of anthocyanins, 3-hydroxyanthocyanidins and 3-deoxyanthoeyanidins in Sorghum bicolor by plasma desorption was developed. Sensitivities to ng levels with minimum sample preparation and analysis were achieved. The technique provides a sensitive means for the measurement of these flavonoid pigments as well as a means to screen for very small quantities of the compounds within plant tissues.

Anion exchange and hydrophobic interaction chromatography of protein preparations extracted in bulk from maize leaves infected with Bipolaris maydis resulted in over a 600-fold purification of 4-hydroxycinnamic acid:CoA ligase. The native enzyme had an estimated molecular weight of about 51·6 kDa, a value consistent with the range of molecular weights reported for the enzyme isolated from dicotyledonous species. Kinetic analyses of enzyme activities with the various phenylpropanoid substrates demonstrated that there was little preferential substrate specificity among p -coumaric, caffeic, and ferulic acids. The enzyme exhibited no activity when assayed with sinapic acid. Previous reports of CoA ligase isoforms in dicotyledonous species have suggested that the enzyme is involved in the diversion of phenylpropanoids to various biosynthetic pathways. However, no evidence for multiple forms was found in the present investigation, suggesting that the maize CoA ligase is not responsible for metabolic channelling of phenylpropanoids and their distribution within the cell which occur as a response to infection. An alternate interpretation to account for the induction of enzyme activity which occurs in response to infection may be based on activation of phenylpropanoids by UDP-glucosyl transferase.

When conidia of Erysiphe graminis are produced, they are hydrophobic and float when placed on the surface of a water droplet. In the hydrophobic state, a conidium may form one or more short germ tubes but fails to form an appressorial germ tube. Formation of an appressorial germ tube, from which the appressorium develops, is required for the establishment of the fungus as a pathogen on leaves of its barley host. Appressorial germ tube formation appears to require conversion of the conidium surface from a hydrophobic to a hydrophilic state. The present investigation shows that this conversion coincides with the contact-stimulated release of a liquid exudate from the conidium. Once the exudate is released, a conidium sinks when placed on water, indicating that it has become relatively more hydrophilic. When hydrophilic conidia are placed on cellophane or the surface of barley leaves, they undergo morphogenesis leading to the formation of appressoria. Incubation of the conidial liquid exudate on the hydrophobic surface of a barley leaf caused the leaf surface to become more hydrophilic. It is suggested that this phenomenon promotes the contact of the conidium with the leaf surface and establishes conditions required for a conidium to germinate by the formation of an appressorial germ tube.

The effects of an elicitor (CG-elicitor) from Colletotrichum graminicola and the host-specific pathotoxin (PC-toxin) produced by Periconia circinata were studied to determine the interactions of responses associated with resistance and susceptibility, respectively. Roots of sorghum ( Sorghum bicolor ) accumulated 3-deoxyanthocyanidin phytoalexins in response to CG-elicitor but not in response to PC-toxin over a range of concentrations. Elicitation of the phytoalexins prior to treatment with PC-toxin had no effect on the genotype-specific induction of electrolyte leakage or on the toxin-enhanced synthesis of a specific group of 16 kDa proteins. Similarly, prior treatment with the elicitor did not prevent infection and development of milo disease symptoms in susceptible seedlings inoculated with conidia of P. circinata . However, treatment of roots with the CG-elicitor enhanced the synthesis of the 16 kDa proteins in both resistant and susceptible genotypes without expression of disease symptoms. Thus, the activities of PC-toxin and CG-elicitor are separable and independent. PC-toxin apparently does not produce disease symptoms by inducing phytoalexins, and induction of phytoalexins does not prevent pathogenesis by P. circinata or the detrimental effects of PC-toxin.

Conidia of Erysiphe graminis f. sp. hordei release a liquid upon contact with a substratum, either the surface of a barley leaf or cellophane. The liquid had been shown to contain esterase activity which partially erodes the surface of the cuticle of barley leaves. The present investigation demonstrates that the liquid exudate contains cutinase activity. The presence of cutinase in the conidial exudate was confirmed by enzyme assays using 3 H-cutin as the substrate. Like other fungal cutinases, the enzyme from E. graminis was inhibited by the serine esterase inhibitor diisopropyl fluorophosphate. The results are significant since they demonstrate for the first time the production of cutinase by a powdery mildew parasite. It is possible that the cutinase is involved in the erosion of the leaf cuticle which begins prior to the development of appressoria. Such erosion may be necessary either for recognition of the host surface by the appressorium or for adhesion and more efficient penetration of the leaf.

On soft substrata conidia of Colletotrichum graminicola germinated by the formation of germ tubes, whereas on hard, hydrophobic substrata they germinated by the formation of appressoria. Regardless of the substratum, conidia did not germinate at high conidium concentrations, suggesting the presence of an endogenous self-inhibitor. Bioassays to test for the inhibition of germination demonstrated the presence of a low-molecular-weight self-inhibitor in the mucilage which surrounds conidia as they are produced in acervuli. The inhibitory material exhibited an absorbance maximum of 310 nm, indicating that it is a member of the mycosporine family of compounds. The inhibitor was purified to homogeneity and shown by mass spectrometric analysis to have a molecular mass consistent with the structure corresponding to mycosporine-alanine.

Microspectrophotometry was performed on intact, pigmented vesicle-like inclusions within living sorghum cells that were accumulating phytoalexins as a response to attempted fungal infection. The results indicate that the deoxyanthocyanidin phytoalexins are present in inclusions. Moreover, the phytoalexin concentration within a single inclusion, based on luteolinidin, was calculated to be 0·15 m . The amounts of luteolinidin and apigeninidin in cells involved in the phytoalexin response at individual infection sites were also determined. The data showed that luteolinidin accumulated to levels of 0·48–1·20 ng/cell whereas apigeninidin accumulated to levels of 0·24–0·91 ng/cell. The results of both analyses confirmed that at the infection site the deoxyanthocyanidins accumulate to levels in substantial excess of those required for inhibition of the fungus Colletotrichum graminicola .

Current understandings and guidelines for sustainable, integrated management of apple diseases are reviewed, and currently effective and sustainable tactics are discussed. Disease management in apples faces several critical problems not seen in other agronomic systems. As long-lived, clonal crops, fungicides and disease resistance play crucial roles in the management of apple diseases. Unfortunately, the pressure placed on these two strategies results in the development of fungicide resistance, and breakdown of host resistance. This review discusses the major diseases of apples, their management strategies, and the problems that have developed to impact sustainable apple production. Symptoms, causal pathogens, disease cycles and management practices are reviewed for primary diseases affecting apples in spring like apple scab, powdery mildew, fire blight and rust diseases. Problems due to fungicide resistance and availability of root stocks and cultivars for exploitation of plant resistance are discussed. Applications of cultural and chemical management with predictive models are also shown. Symptoms, disease cycles and management are also reviewed for summer diseases of apple, like bitter rot, flyspeck and sooty blotch

Extracts of conidia and the conidial mucilage of Colletotrichum graminicola contained materials that elicited the accumulation of deoxyanthocyanidin phytoalexins in sorghum mesocotyls and juvenile sorghum leaves. Elicitor activity was associated with peptide and carbohydrate components extracted from conidia and with peptides removed from conidia by repeated washing. The accumulation in mesocotyl tissue of deoxyanthocyanidin phytoalexins occurred linearly over time in response to a single elicitor concentration and the amount of deoxyanthocyanidins that accumulated also increased linearly in response to increasing concentrations of crude elicitor preparations. Elicitor activity was highly specific for sorghum in that crude preparations did not elicit a visible response from nine other plant species. Neither wounding by cutting nor gentle abrasion of the sorghum tissue was sufficient to elicit the accumulation of the deoxyanthocyanidin phytoalexins.

Contact with a surface stimulates the release of a liquid from conidia of Erysiphe graminis and the liquid has been shown to contain esterase activity. The present investigation demonstrates that the liquid exudate partially erodes the cuticle of barley leaves. The release of esterase into the liquid occurs in two stages. The second stage of release occurs between 10 and 15 min after contact and was shown to be temperature dependent and to be inhibited by cycloheximide. Cuticular erosion may provide a stimulus for subsequent morphological differentiation, such as appressorium formation, or it may enhance appressorial adhesion.

The accumulation of a new 3-deoxyanthocyanidin phytoalexin in sorghum leaves and mesocotyls was demonstrated to occur following inoculation with an isolate of Colletotrichum graminicola pathogenic to sorghum, an isolate of C. graminicola pathogenic only to corn, and an isolate of Helminthosporum maydis (a nonpathogen of sorghum). The compound was found to be a caffeic acid ester of arabinosyl-5-O-apigeninidin and was never present in uninoculated tissues. Comparison of the fungitoxicity of the compound with that of two phytoalexins identified previously (apigeninidin and luteolinidin) showed that all three of the compounds exhibited toxicity to C. graminicola at concentrations of less than 10 μ m .

Corn leaves inoculated with the fungal pathogens Colletotrichum graminicola or Helminthosporium maydis accumulated two caffeoyl esters that were extractable. These were identified as caffeoyl glucose, and a caffeoyl ester with an unknown acidic moiety. The response was not cultivar specific as a number of susceptible and resistant cultivars produced both compounds following infection. Increased levels of the two esters were detected at infection sites 20 hr after inoculation when visible symptoms of disease first became apparent as minute necrotic flecks. The manner in which the two compounds accumulated and the fact that they were not inhibitory in vitro to either pathogen may indicate that their function in the disease response involves host metabolism rather than fungal inhibition.