Adhesion Of Conidia Research Articles

Analysis of a Blumeria graminis-Secreted Lipase Reveals the Importance of Host Epicuticular Wax Components for Fungal Adhesion and Development

The biotrophic powdery mildew fungus Blumeria graminis releases extracellular materials to the surface of fungal infection structures that facilitate anchoring them to hydrophobic plant surfaces prior to infection; however, the chemistry of fungal adhesives and the mechanism of adhesion remain largely unclear. Expressed sequence tag analysis led to identification of a secreted lipase, Lip1, from B. graminis. Expression of LIP1 is dramatically upregulated during the early stages of fungal development. Lip1, secreted to the surface of fungal cell walls, possesses lipolytic activity against a broad range of glycerides and releases alkanes and primary fatty alcohols from the epicuticular wax of wheat leaves. Of the epicuticular wax components released by Lip1 activity, long-chain alkanes are the most efficient cues for triggering appressorium formation. Pretreatment of wheat leaves with Lip1, thereby removing leaf surface wax, severely compromises components of fungal pathogenicity, including conidial adhesion, appressorium formation, and secondary hypha growth. Our data suggest that Lip1 activity releases cues from the host surface to promote pathogen development and infection.

A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial hydrophobicity when expressed in Beauveria bassiana

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.

MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae

Fasciclin family proteins have been identified as cell adhesion molecules in various organisms. In this study, a novel Magnaporthe oryzae fasciclin-like protein encoding gene, named MoFLP1, was isolated from a subtractive suppressive cDNA library and functionally analyzed. Sequence analysis showed that the MoFLP1 gene contains an open reading frame (ORF) of 1050 nucleotides encoding 349 amino acids with a calculated molecular weight of 35.85 kDa and a pI of 7.76. The deduced MoFLP1 protein contains a 17-amino acid secretion signal sequence and an 18-amino acid sequence with the characteristics of a glycosylphosphotidylinositol (GPI) anchor additional signal at its N- and C-terminuses, respectively. Potential N-glycosylation sites and domains involving cell adhesion were also identified in MoFLP1. Sequence analysis and subcellular localization by the expression of MoFLP1-GFP fusion construct in M. oryzae indicated that the MoFLP1 protein is probably localized on the vacuole membrane. Two MoFLP1 null mutants generated by targeted gene disruption exhibited marked reduction of conidiation, conidial adhesion, appressorium turgor, and pathogenicity. Our results indicate that fasciclin proteins play important roles in fungal development and pathogenicity in M. oryzae.

Estudos histoplásticos da interação Colletotrichum gloeosporioides: cafeeiro

Nos estudos envolvendo a interação Colletotrichum gloeosporioides-cafeeiro (Coffea arabica L.), poucas são as informações a respeito do modo de penetração e colonização deste patógeno. Estudou-se por meio da microscopia eletrônica de varredura (MEV), os eventos de pré-penetração e penetração de C. gloeosporioides em hipocótilos de cafeeiros e a colonização natural de ramos e frutos provenientes de plantas com mancha manteigosa. Realizaram-se três ensaios. No primeiro, fez-se a observação dos eventos de pré-penetração de C. gloeosporioides inoculados em hipocótilos de plântulas da cv. Acaiá Cerrado com e sem ferimentos; no segundo, observou-se a colonização de ramos enfermos em que havia murcha drástica e necrose local e, no terceiro, a colonização de frutos enfermos. A germinação dos conídios nos hipocótilos feridos ocorreu 6 horas após a inoculação, com a formação de um ou dois tubos germinativos terminais e a adesão dos conídios nas depressões dos hipocótilos. Apressórios globosos a subglobosos e de contorno regular surgiram 12 horas após a inoculação. Até 72 horas, não foi possível observar a formação de acérvulos sobre os tecidos submetidos à inoculação. Nos ramos observava-se colonização nos vasos floema e parênquima cortical. Nos frutos em diferentes estádios de maturação foi observada a colonização do tecido próximo à epiderme e colapso dos estômatos na área lesionada.

Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae

Metarhizium anisopliae is a model system for studying insect fungal pathogenesis. The role of cAMP signal transduction in virulence was studied by disrupting the class I PKA catalytic subunit gene ( MaPKA1). The PKA mutant ( ΔMaPKA1) showed reduced growth and greatly reduced virulence. PKA was dispensable for differentiation of infection structures (appressoria), but differentiation was delayed and the appressoria were defective because of reduced turgor pressure. ΔMaPKA1 germinated at similar rates as the wild type in glucose and glycerol, but germination was delayed on alanine. Conidial adhesion and appressorium formation by ΔMaPKA1 against a plastic surface was fully inhibited with glucose as sole nutrient source. Adhesion to plastic was not inhibited with glycerol or alanine, but appressorium formation was delayed. ΔMaPKA1 showed reduced tolerance to the oxidative agent diamide, but not to H 2O 2 and methyl-viologen. Comparative transcriptome analysis of ΔMaPKA1 and the wild type strain showed that PKA is responsible for up-regulating approximately one-third of the genes induced by insect cuticle, including subsets of those responsible for differentiation of appressoria and penetration pegs, cuticle degradation, nutrient acquisition, pH regulation, lipid synthesis, cell cycle control and the cytoskeleton. PKA was not however required for expression of toxin-producing genes. We conclude therefore that MaPKA1 is required for sensing host-related stimuli and transduction of these signals to regulate many infection processes.

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.

Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus

Growth under stress may influence pathogen virulence and other phenotypic traits. Conidia of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae (isolate ARSEF 2575) were produced under different stress conditions and then examined for influences on in vitro conidial germination speed, adhesion to the insect cuticle, and virulence to an insect host, Tenebrio molitor. Conidia were produced under non-stress conditions [on potato–dextrose agar plus 1 g l −1 yeast extract (PDAY; control)], or under the following stress conditions: osmotic (PDAY + sodium chloride or potassium chloride, 0.6 or 0.8 m); oxidative [(PDAY + hydrogen peroxide, 5 m m) or UV-A (irradiation of mycelium on PDAY)]; heat shock (heat treatment of mycelium on PDAY at 45 °C, 40 min); and nutritive [minimal medium (MM) with no carbon source, or on MM plus 3 g l −1 lactose (MML)]. Conidia were most virulent (based on mortality at 3 d) and had the fastest germination rates when produced on MML, followed by MM. In addition, conidial adhesion to host cuticle was greatest when the conidia were produced on MML. Media with high osmolarity (0.8 m) produced conidia with slightly elevated virulence and faster germination rates than conidia produced on the control medium (PDAY), but this trend did not hold for media with the lower osmolarity, (0.6 m). Conidia produced from mycelium irradiated with UV-A while growing on PDAY had somewhat elevated virulence levels similar to that of conidia produced on MM, but their germination rate was not increased. Hydrogen peroxide and heat shock treatments did not alter virulence. These results demonstrate that the germination, adhesion and virulence of M. anisopliae conidia can be strongly influenced by culture conditions (including stresses) during production of the conidia.

Influence of additives on adhesion of Penicillium frequentans conidia to peach fruit surfaces and relationship to the biocontrol of brown rot caused by Monilinia laxa

Additives, such as sucrose, d-sorbitol, glycerol, sodium alginate, carboxymethyl cellulose, silica gel, gelatine, non-fat skimmed milk and a commercial adhesive were added to conidia of Penicillium frequentans at two different points in the production process of the formulation of this fungus to improve conidial adhesion. Conidial adhesion was estimated as the number of P. frequentans conidia (no. conidia cm − 2 ) and colony-forming units of P. frequentans per unit area (cfu cm − 2 ) that adhered to glass slides or to peach surfaces. The P. frequentans conidial concentration had a significant effect on conidial adhesion, while the shelf life of conidia did not have any effect. The highest adhesion of P. frequentans conidia to glass slides was observed when conidial concentrations were greater than 10 6 conidia ml − 1 . P. frequentans conidial adhesion was improved when 1.5% sodium alginate or 1.5% carboxymethyl cellulose were added to the conidial mass obtained after production and before drying by the fluid bed drying process. Conidial adhesion was also enhanced when 1.5% sodium alginate, 1.5% carboxymethyl, or 1.5% gelatine were added to conidia after fluid bed drying. P. frequentans formulations with 1.5% sodium alginate or 1.5% carboxymethyl cellulose were more effective in reducing brown rot caused by Monilinia laxa than dried P. frequentans conidia alone. Our results show that additives can improve adhesion of P. frequentans conidia to fruit surfaces, resulting in more effective control of brown rot in peaches.

Adhesion of conidia and germlings of the plant pathogenic fungus Bipolaris sorokiniana to solid surfaces

Soon after coming in contact with its host, the plant pathogenic fungus Bipolaris sorokiniana produces an extracellular material that appears to be important for adhering conidia and germlings to the host surface. To further understand this step of the infection, the adhesion of B. sorokiniana to artificial solid surfaces was examined. On a hydrophobic (polystyrene) surface adhesion occurred in two stages, the first by conidia and the second by germlings. Conidial adhesion occurred shortly (0-1 h) after hydration. The conidia were easily detached by increasing the shear force and including detergents in the washing buffer. As conidia were hydrophobic, these observations indicate that conidial adhesion to polystyrene is due to weak, hydrophobic interaction. The second stage of adhesion was accompanied by conidial germination and occurred 1-2 h after hydration and contact with the surface. Concomitant with the delayed adhesion, the fungus produced an extracellular matrix (ECM). The adhesion of germlings was firm and surface-unspecific since they adhered to both hydrophobic and hydrophilic (glass) surfaces, Except for strong bases, hydrochloric acid and broad-specificity proteases (including Pronase E), none of the hydrolytic enzymes, electrolyte solutions, ionic and hydrophobic detergents and organic solvents removed germlings from the solid surfaces. The adhesion of germlings incubated in the presence of the protein glycosylation inhibitor tunicamycin or the lectins Con A (Concanavalin A) and GNA (from Galanthus nivalis) was significantly reduced, which indicates the involvement of surface glycoproteins in this process. The surface proteins of germlings were labelled with I-125, extracted and analysed by two-dimensional gel electrophoresis. This revealed about 40 surface proteins over a wide pH range (4-10) with molecular masses between 10 and 100 kDa.

Recognition of laminin byParacoccidioides brasiliensisconidia: a possible mechanism of adherence to human type II alveolar cells

This study addresses the recognition of laminin by Paracoccidioides brasiliensis conidia, as well as its possible role in the adherence of conidia to A549 cells. Adherence of conidia to immobilized laminin was shown to be specific, as anti-laminin antibodies, soluble laminin or the laminin-derived peptides IKVAV and CDPGYIGSR inhibited this interaction. RGD containing peptides and various monosaccharides had no effect on adherence, with the exception of N-acetylneuraminic acid. Pre-treatment of conidia with fibrinogen and fibronectin, but not with BSA, also resulted in significant inhibition, suggesting that P. brasiliensis conidia might cross-recognize host proteins involved in colonization. In assays using transmission electron microscopy, we observed internalization of conidia 30 min after exposition to A549 cells. Laminin present on the surface of A549 cells shown to serve as mediator of this interaction, with a significant decrease in fungal adherence when the epithelial cells were pre-treated with anti-laminin antibodies or when conidia were pre-incubated with either soluble laminin or the laminin-specific peptides. Together these results suggest that the recognition of laminin by P. brasiliensis conidia is a key process in the interaction with pulmonary epithelial cells, where this extracellular matrix protein acts as bridging molecule.

Regulation and role of a STE12‐like transcription factor from the plant pathogen Colletotrichum lindemuthianum

In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. However, their regulation and downstream targets are still unknown. In the present study, a STE12-like gene (CLSTE12) from Colletotrichum lindemuthianum was isolated. We identified a spliced variant whose expression was negatively regulated during early stages of pathogenesis, whereas the correctly spliced mRNA remained expressed up to the penetration step, suggesting distinct roles for these two transcripts. Indeed, the full-length sequence was able to complement a yeast STE12 mutant, whereas overexpression of the transcript variant had a dominant-negative effect on yeast invasive growth and C. lindemuthianum pathogenicity. To further investigate the downstream genes that could be regulated by CLSTE12, disruption mutants were generated. Phenotypic analyses of the mutants revealed reduced pectinase activity and conidial adhesion to polystyrene. Analysis of cell surface proteins allowed the identification of a major protein, Clsp1p, which was absent from the mutants. Clsp1p belongs to a new family of wall-associated proteins only found in euascomycetous fungi. Overall, these results suggest that the activity of CLSTE12 can be modulated by a regulated alternative splicing mechanism and that this factor is involved in the production of cell surface proteins and host cell wall degrading enzymes.

Chondroitin sulfate B and heparin mediate adhesion of Penicillium marneffei conidia to host extracellular matrices

Penicilliosis is a disseminated infection in immunocompromised individuals caused by the dimorphic fungus, Penicillium marneffei. Very little is known about its route of infection, however, it is thought that initial infection occurs through inhalation of conidia. We investigated the role played by various extracellular matrix glycosaminoglycans (GAGs) in the initial adherence of P. marneffei conidia using a direct adhesion assay. GAGs were further used to block the binding of fungal spores to human lung epithelial cells and highly sulfated GAGs were tested for their inhibitory effects owing to their degree of sulfation. Our results demonstrated high levels of conidial adhesion to chondroitin sulfate B, heparin and highly sulfated chitosan (CP-3). No direct adherence was observed to immobilized chondroitin sulfate (CS) A, CSC, CSD and hyaluronic acid, as well as chitosans with low sulfate content. The results suggested that P. marneffei conidia bind to iduronic acid (IdoA) of the polysaccharide chains. Involvement of negatively charged sulfate groups in adhesion was also indicated. Furthermore, significant inhibition of conidial adherence to A549 cells was observed in the presence of CSB, heparan sulfate (HS), heparin and CP-3. It was further demonstrated that GAGs can affect the adhesion of conidia to fibronectin and laminin, glycoproteins that have previously been implicated as adhesive receptors for fungal conidia. CSB and HS could partially inhibit the adhesion of fungal conidia to laminin and fibronectin implying that conidia can weakly interact with the IdoA GAG-binding domain(s) of these molecules. The data indicated that, in addition to fibronectin and laminin, IdoA-containing GAGs may play an important role in fungal adherence to the surface of human lung epithelium.

Evaluation of novel fungal and nematode isolates for control of Conotrachelus nenuphar (Coleoptera: Curculionidae) larvae

Abstract The primary objective was to identify potential nematode and fungus pathogens for control of plum curculio (Conotrachelus nenuphar) larvae. Initial bioassays were conducted in the laboratory. Seventeen isolates (13 wild-types and four color mutants) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae were screened. Seven of the isolates were highly virulent for plum curculio larvae (LT50 = 4.0–5.4 days at a concentration of 107 conidia ml−1). Of these isolates, four were color mutants induced by UV-B irradiation exposure (DWR 180, 145, 142, and 62), and three of these showed significantly higher virulence than their parental wild-types (ARSEF 2575 and 23). The most virulent wild-type was the isolate ARSEF 1187. Surprisingly, higher virulence was not correlated with higher conidial adhesion to the cuticle of plum curculio larvae. A Utah-collected isolate of Heterorhabditis bacteriophora was as effective as a commercial strain of Steinernema feltiae in killing plum curculio larvae in laboratory bioassays. Larval mortality ranged from 21 to 89% at inoculum concentrations of 0.125 to 4.0 × 106 infective juveniles (IJs) per m−2. A diapausing (northern) population of plum curculio was less susceptible to S. feltiae (LC50 = 8.6 × 105 IJs m−2) than a non-diapausing (southern) population (LC50 = 3.6 × 105 IJs m−2). A time delay in adding plum curculio larvae to sterilized soil treated with S. feltiae significantly reduced insect mortality after 2 days and reduced mortality below 50% after a delay of 7 days. In field tests, S. feltiae killed 22–39% of northern plum curculio larvae at concentrations of 0.5–2.0 × 106 IJs m−2.

A Proposed Role for the Cuticular Fatty Amides of Liposcelis bostrychophila (Psocoptera: Liposcelidae) in Preventing Adhesion of Entomopathogenic Fungi with Dry-conidia

Maximum challenge exposure of Liposcelis bostrychophila to Beauveria bassiana, Paecilomyces fumosoroseus, Aspergillus parasiticus or Metarhizium anisopliae resulted in no more than 16% mortality. We investigated several of L. bostrychophila's cuticular lipids for possible contributions to its tolerance for entomopathogenic fungi. Saturated C14 and C16 fatty acids did not reduce the germination rates of B. bassiana or M. anisopliae conidia. Saturated C6 to C12 fatty acids that have not been identified in L. bostrychophila cuticular extracts significantly reduced germination, but the reduction was mitigated by the presence of stearamide. Cis-6-hexadecenal did not affect germination rates. Mycelial growth of either fungal species did not occur in the presence of caprylic acid, was reduced by the presence of lauric acid, and was not significantly affected by palmitic acid. Liposcelis bostrychophila is the only insect for which fatty acid amides have been identified as cuticular components. Stearamide, its major fatty amide, did not reduce germination of B. bassiana or M. anisopliae conidia or growth of their mycelia. Adhesion of conidia to stearamide preparations did not differ significantly from adhesion to the cuticle of L. bostrychophila. Pretreatment of a beetle known to be fungus-susceptible, larval Oryzaephilus surinamensis, with stearamide significantly decreased adhesion of B. bassiana or M. anisopliae conidia to their cuticles. This evidence indicates that cuticular fatty amides may contribute to L. bostrychophila's tolerance for entomopathogenic fungi by decreasing hydrophobicity and static charge, thereby reducing conidial adhesion.

Adhesion of decay-causing fungal conidia in wounds ofMalus×domestica'Golden Delicious' apple fruit is influenced by wound age

Wounds are the primary site in apple fruit for infection by conidia of Botrytis cinerea Pers.:Fr. and Penicil lium expansum Link & Thom. The effects of wound shape, wound age, and chemical properties of the wound on conidial adhesion in wounds of Malus ×domestica Borkh. 'Golden Delicious' fruit were investigated. Adhesion was measured after dislodging conidia from wounds using a sonication probe above the wound. In all experiments, conidial adhesion responses were similar for both fungi. Conidial adhesion in puncture wounds was not different from adhesion in slice wounds. Wound age, however, profoundly affected conidial adhesion. Conidia of both fungi exhibited 78.1%–91.9% adhesion in freshly made wounds of both shapes compared with 37.7%–56.6% in 1-d-old wounds. Conidial adhesion increased as wound age increased from 1 to 5 d. Exposure of 1- and 2-d-old wounds to butyl acetate, a volatile constituent of apple fruit, increased conidial adhesion compared with nonexposed wounds. This finding, in addition to results from the histochemical analyses of wounds, the quantification of sugars and total phenolics in water diffusates from wounds, and the measurement of conidial adhesion to wound diffusates, suggested that conidial adhesion in wounds was influenced by altered surface chemistry of wounds as they aged.Key words: apple fruit wounds, decay-causing fungi, fungal spore adhesion, mycoactive acetate esters, wound aging, wound decay.

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We isolated an entomopathogenic fungus, Beauveria amorpha (Strain: HpBa-1), from Heptophylla picea larva. When the larvae were inoculated with HpBa-1, 90.0% cumulative mortality was obtained in 30 days. However, the cumulative mortality was low when the larvae were inoculated with Metarhizium anisopliae (Strain: PMA-7) isolated from H. picea larvae. The high virulence of HpBa-1 to H. picea larvae was analyzed by investigating the conidial adhesion and germination of HpBa-1 on larval cuticle. Numerous HpBa-1 conidia adhered to the H. picea cuticle and most conidia on the H. picea cuticle remained over time. HpBa-1 conidia germinated on H. picea cuticle 96 h postinoculation. However, few PMA-7 conidia adhered to the H. picea cuticle as compared to HpBa-1 and did not germinate on the cuticle 96 h postinoculation. From these results, conidial adhesion and germination on the cuticle of H. picea is one factor causing high virulence of HpBa-1 to the larvae.

Germination and adhesion of fungal conidia on polycarbonate membranes and on apple fruit exposed to mycoactive acetate esters.

The adhesion and germination of conidia of nine fungal species were assessed on polycarbonate membranes or on the skin of apple fruit in sealed glass bottles injected or not injected with acetate esters. Adhesion was determined after dislodging conidia from surfaces using a sonication probe. Adhesion and germination of conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Penicillium citrinum, Penicillium claviforme, or Trichoderma sp. on membranes after 48 h were not increased in a 1.84 microg mL(-1) headspace of butyl acetate (BA), ethyl acetate, hexyl acetate, 2-methylbutyl acetate, pentyl acetate, or propyl acetate. Adhesion and germination of Botrytis cinerea, Penicillium expansum, and Penicillium roquefortii conidia were stimulated by all esters. Only conidia of B. cinerea and P. expansum exhibited increased adhesion and germination on the skin of apple fruit in bottles exposed to 0.92 microg mL(-1) of BA. Only conidia of B. cinerea and P. expansum produced decay in inoculated puncture wounds on fruit. Freshly made puncture wounds or 24-h-old puncture wounds in fruit were more adhesive than the unpunctured skin of fruit to conidia of B. cinerea or P. expansum. Fresh wounds were more adhesive to both fungi than 24-h-old puncture wounds. The skin and wounds of fruit were as adhesive to B. cinerea conidia as they were to P. expansum conidia. A 4-h exposure to 1.43 microg mL(-1) of BA increased adhesion of B. cinerea and P. expansum conidia in 24-h-old wounds. Results suggest that acetate-ester stimulation most likely is not a rare phenomenon in the fungi. For nutrient-dependent decay pathogens of apple fruit, acetate esters may be an alternative chemical cue used to maintain adhesion of conidia to wound surfaces.

Ontogenic Resistance and Plant Disease Management: A Case Study of Grape Powdery Mildew

ABSTRACT A fundamental principle of integrated pest management is that actions taken to manage disease should be commensurate with the risk of infection and loss. One of the less-studied factors that determines this risk is ontogenic, or age-related resistance of the host. Ontogenic resistance may operate at the whole plant level or in specific organs or tissues. Until recently, grape berries were thought to remain susceptible to powdery mildew (Uncinula necator) until late in their development. However, the development of ontogenic resistance is actually quite rapid in berries, and fruit become nearly immune to infection within 4 weeks after fruit set. Our objective was to determine how and at what stage the pathogen was halted in the infection process on ontogenically resistant berries. Adhesion of conidia, germination, and appressorium formation were not impeded on older berries. However, once berries were approximately 3 weeks old and older, few germlings were able to form secondary hyphae. Ontogenically resistant berries responded rapidly to infection by synthesis of a germin-like protein that had been previously shown to play a role in host defense against barley powdery mildew. On susceptible berries, cell discoloration around penetration sites indicated the oxidation of phenolic compounds; a process that was followed by localized cell death. However, the pathogen was still able to infect such cells prior to their death, continue secondary growth, and thereby colonize young berries. Formation of papillae was not involved in the differential resistance mechanism of older berries. In susceptible berries, papillae formed frequently at infection sites but did not always contain the pathogen, whereas in resistant berries, the pathogen was always halted prior to the formation of papillae. The host defense, which conditions ontogenic resistance, operates in the earliest stages of the infection process, in the absence of gross anatomical barriers, prior to the formation of a functional haustorium and prior to the development of a conspicuous penetration pore. We also found that diffuse powdery mildew colonies that were not visible in the field predisposed berries to bunch rot by Botrytis cinerea, increased the levels of infestation by spoilage microorganisms, and substantially degraded wine quality. Our improved understanding of the nature, causes, and stability of ontogenic resistance in the grapevine/ powdery mildew system has supported substantial changes in how fungicides are used to control the disease. Present applications are more focused on the period of maximum fruit susceptibility instead of following a calendar-based schedule. This has improved control, reduced losses, and in many cases reduced the number of fungicide applications required to suppress the disease. Particularly where fungicides are deployed in a programmatic fashion and ontogenic resistance is dynamic, there may be equivalent improvements to be made in other hostpathogen systems through studies of how host susceptibility changes through time.

Mycoactive acetate esters from apple fruit stimulate adhesion and germination of conidia of the gray mold fungus.

Ethyl acetate, 2-methylbutyl acetate, butyl acetate (BA), and hexyl acetate were detected by solid-phase microextraction and gas-liquid chromatography inside slices of Golden Delicious apple and in water droplets on the skin of slices incubated in sealed glass jars. Conidial adhesion and germination of the gray mold fungus, Botrytis cinerea, was assessed on apple slices after exposure or no exposure to the esters in the headspaces of glass jars. Attached conidia were dislodged by sonication and remaining conidia on apple slices were counted by microscopy. Adhesion generally increased as BA increased to 7.2 microg mL(-1), but declined with greater concentrations. BA at 0-3.6 microg mL(-1) for 24 h stimulated adhesion 2-fold greater compared to that at 4 h. Adhesion stimulated by BA increased as a function of time (0-24 h), showing linear trends (r (2) = 0.99; p = 0.01) during 0-12 h. The four esters were similar in their ability to stimulate adhesion. Germination of conidia exposed to BA increased linearly (r (2) = 0.95-0.98; p = 0.01) during 4-12 h. Conidial adhesion stimulated by BA preceded conidial germination by 2 h. The four esters stimulated conidial germination to similar levels. Results indicated that acetate esters formed in apple fruit are mycoactive, influencing life-cycle events of B. cinerea important to its survival on the fruit. The similar responses of three B. cinerea isolates to four acetate esters suggests a common stimulation mechanism may operate in B. cinerea.

Adhesion of conidia and germlings of the plant pathogenic fungus Bipolaris sorokiniana to solid surfaces

Soon after coming in contact with its host, the plant pathogenic fungus Bipolaris sorokiniana produces an extracellular material that appears to be important for adhering conidia and germlings to the host surface. To further understand this step of the infection, the adhesion of B. sorokiniana to artificial solid surfaces was examined. On a hydrophobic (polystyrene) surface adhesion occurred in two stages, the first by conidia and the second by germlings. Conidial adhesion occurred shortly (0-1 h) after hydration. The conidia were easily detached by increasing the shear force and including detergents in the washing buffer. As conidia were hydrophobic, these observations indicate that conidial adhesion to polystyrene is due to weak, hydrophobic interaction. The second stage of adhesion was accompanied by conidial germination and occurred 1-2 h after hydration and contact with the surface. Concomitant with the delayed adhesion, the fungus produced an extracellular matrix (ECM). The adhesion of germlings was firm and surface-unspecific since they adhered to both hydrophobic and hydrophilic (glass) surfaces. Except for strong bases, hydrochloric acid and broad-specificity proteases (including Pronase E), none of the hydrolytic enzymes, electrolyte solutions, ionic and hydrophobic detergents and organic solvents removed germlings from the solid surfaces. The adhesion of germlings incubated in the presence of the protein glycosylation inhibitor tunicamycin or the lectins Con A (Concanavalin A) and GNA (from Galanthus nivalis ) was significantly reduced, which indicates the involvement of surface glycoproteins in this process. The surface proteins of germlings were labelled with 125 I, extracted and analysed by two-dimensional gel electrophoresis. This revealed about 40 surface proteins over a wide pH range (4-10) with molecular masses between 10 and 100 kDa.