F. M. Dewey

The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea

Fruit ripening is characterized by processes that modify texture and flavor but also by a dramatic increase in susceptibility to necrotrophic pathogens, such as Botrytis cinerea. Disassembly of the major structural polysaccharides of the cell wall (CW) is a significant process associated with ripening and contributes to fruit softening. In tomato, polygalacturonase (PG) and expansin (Exp) are among the CW proteins that cooperatively participate in ripening-associated CW disassembly. To determine whether endogenous CW disassembly influences the ripening-regulated increase in necrotropic pathogen susceptibility, B. cinerea susceptibility was assessed in transgenic fruit with suppressed polygalacturonase (LePG) and expansin (LeExp1) expression. Suppression of either LePG or LeExp1 alone did not reduce susceptibility but simultaneous suppression of both dramatically reduced the susceptibility of ripening fruit to B. cinerea, as measured by fungal biomass accumulation and by macerating lesion development. These results demonstrate that altering endogenous plant CW disassembly during ripening influences the course of infection by B. cinerea, perhaps by changing the structure or the accessibility of CW substrates to pathogen CW-degrading enzymes. Recognition of the role of ripening-associated CW metabolism in postharvest pathogen susceptibility may be useful in the design and development of strategies to limit pathogen losses during fruit storage, handling, and distribution.

Identification and localization of an active cutinase in the pollen of Brassica napus L.

Polyclonal antiserum and monoclonal antibodies raised to a purified cutinase from Fusarium solani f. sp. pisi have been used to identify an active cutinase in the pollen of Brassica napus. These antibodies recognized a polypeptide with an estimated molecular weight of 22kDa — a molecular weight indentical to that of the Fusarium cutinase — and localized this polypeptide to the intine of the pollen wall. Enzyme assays on the renatured 22kDa polypeptide after electroelution from a preparative SDS-PAGE gel revealed the polypeptide to be an enzyme capable of catalysing the hydrolysis of tritiated apple cutin and the synthetic substrate p-nitrophenyl butyrate. The molecular weight, immunological properties and substrate specificity of the Brassica cutinase suggest that this enzyme resembles more closely fungal cutinases than it does the cutinase from the pollen of Nasturtium (Tropaeolum majus) — the only angiosperm cutinase so far characterized (Maiti et al., 1979, Arch. Biochem. Biophys. 196, 412–423). These differences between the pollen cutinases from two members of the Dicotyledoneae are unexpected and predict a diversity of this class of pollen enzyme within the angiosperms.

Infection mechanisms of Botrytis species: pre-penetration and pre-infection processes of dry and wet conidia

Light and electron microscope studies of the infection mechanisms of two species of Botrytis, B. cinerea and B. fabae, axe described. Conidia were inoculated dry or in the presence of aqueous glucose onto glass and Vicia faba leaves and incubated in a moist chamber. In all cases, dry-inoculated conidia germinated rapidly to produce short germ-tubes only. In contrast, in the presence of aqueous glucose, conidia germinated to produce long germ-tubes which extended across the substratum. Low temperature SEM of B. cinerea conidia confirmed these results and showed further that in the presence of aqueous glucose, the germ-tubes were enclosed by an extensive sheath of fibrillar-like material. No such matrix material was found to be associated with the conidia or germ-tubes of dry-inoculated conidia. Immunofluorescence microscopy and immunogold labelling of ultrathin sections of B. fabae-infected bean leaves were carried out using the Botrytis-specific monoclonal antibody, BC-KH4. In every case antibody-binding was observed at the surface of both dry and wet conidia. Furthermore, when conidia were inoculated in the presence of aqueous glucose, antibody-binding was also located throughout the fibrillar-like matrix material associated with conidia and germ-tubes. At the TEM level, traces of amorphous matrix material were found to be associated with the short germ-tubes of dry-inoculated conidia at the site of penetration of the leaf surface. This amorphous matrix material also immunolabelled with BC-KH4. Low-temperature SEM of the macroscopic lesions formed on infection of bean leaves by Botrytis species also revealed that these lesions corresponded to clearly defined areas of collapsed epidermal cells.

Development of monoclonal-antibody-ELISA, -DOT-BLOT and -DIP-STICK immunoassays for Humicola lanuginosa in rice

Monoclonal antibodies (MAbs) were raised against Humicola lanuginosa, a thermophilic, saprophytic fungus that is commonly isolated from freshly harvested rice grains in Indonesia. Mice were immunized by direct injection into the peritoneum, without prior concentration, of fresh cell-free surface washings from a solid agar slant culture. Hybridoma supernatants were screened by ELISA using wells coated with a dilution of the immunogen. From one fusion 403 hybridoma clones were obtained yielding 52 cell lines secreting antibodies positive for H. lanuginosa. Twelve cell lines were re-cloned, grown in bulk and tested against other storage fungi. Most of the MAbs raised were IgM antibodies that cross-reacted with several of the storage fungi and/or uninfected rice grains. However, the IgM antibody EC6 did not recognize antigens from rice grains and cross-reacted strongly with only one other test fungus, Penicillium variabile, and partially with two others. This MAb was used to develop a highly sensitive DIP-STICK immunoassay to detect the fungus in infected grains. These assays are simple to perform, require no equipment and are suitable for field use by untrained workers.

Development of monoclonal-antibody-ELISA and DIP-STICK immunoassays for Penicillium islandicum in rice grains.

Monoclonal antibodies (mAbs) were raised against Penicillium islandicum, a fungus commonly isolated from stored rice grains in South-East Asia. Mice were immunized by a direct, simple method; fresh cell-free surface washings from a solid agar slant culture were injected directly into the peritoneum without prior concentration. Hybridoma supernatants were screened by ELISA. Most of the mAbs raised cross-reacted with other storage fungi and/or uninfected rice grains but there were species-specific. One of these, PI01, was used to develop ELISA and DIP-STICK assays for the detection of P. islandicum in individual grains. All inoculated grains and approximately 90% of grains in natural infected samples from Indonesia tested positively, by ELISA, for P. islandicum. This result and those obtained for discoloured grains from both Indonesia and the Philippines, 32% and 14% respectively, are higher than those obtained by direct plating of surface-sterilized grains. Heat and periodate treatment of the PI01 antigen and binding on Western blots indicate that it is a glycoprotein of Mr greater than 90,000. Hyphae of all ages stained uniformly by immunofluorescence using the PI01 antibody but mature conidia stained only weakly.

A new method to monitor airborne inoculum of the fungal plant pathogens Mycosphaerella brassicicola and Botrytis cinerea.

ABSTRACT We describe a new microtiter immunospore trapping device (MTIST device) that uses a suction system to directly trap air particulates by impaction in microtiter wells. This device can be used for rapid detection and immunoquantification of ascospores ofMycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores ofM. brassicicola correlation coefficients (r2) of 0.943 and 0.9514 were observed for the number of MTIST device-impacted ascospores per microtiter well and the absorbance values determined by ELISA, respectively. These values were not affected when a mixed fungal spore population was used. There was a relationship between the number of MTIST device-trapped ascospores of M. brassicicola per liter of air sampled and the amount of disease expressed on exposed plants ofBrassica oleracea (Brussels sprouts). Similarly, when the MTIST device was used to trap conidia of B. cinerea, a correlation coefficient of 0.8797 was obtained for the absorbance values generated by the ELISA and the observed number of conidia per microtiter well. The relative collection efficiency of the MTIST device in controlled plant growth chambers with limited airflow was 1.7 times greater than the relative collection efficiency of a Burkard 7-day volumetric spore trap for collection of M. brassicicola ascospores. The MTIST device can be used to rapidly differentiate, determine, and accurately quantify target organisms in a microflora. The MTIST device is a portable, robust, inexpensive system that can be used to perform multiple tests in a single sampling period, and it should be useful for monitoring airborne particulates and microorganisms in a range of environments.

Efficacy of different immunogens for raising monoclonal antibodies to Botrytis cinerea.

A panel of monoclonal antibodies (MAbs) was raised to Botrytis cinerea using four different immunogens. Co-immunization with cross-reactive monoclonal antibodies helped to increase the percentage of specific hybridoma cell lines produced. The abilities of the MAbs to detect fungal antigens in extracts from infected tissue was correlated with their taxonomic specificities and with the localization of the antigens on the surfaces of the hyphae and conidia. Four distinct groups were identified: taxonomically unrelated, near-genus-, genus- and near-isolate-specific. Genus-specific antibodies gave the highest absorbance values in ELISAs with extracts from infected plant tissues. MAbs from this group all recognized heat-stable epitopes on antigens expressed along the length of the extracellular matrix of the hyphae and surface of the conidia. Antibodies from this group were all IgG1 antibodies.

Use of Monoclonal Antibodies to Detect, Quantify and Visualize Fungi in Soils

Publisher Summary This chapter discusses the application of immunological methods for the detection, quantification and visualization of soil-borne fungi using monoclonal antibodies (MAbs). Fungal MAbs recognize higher molecular weight glycoprotein molecules that appear to be abundant throughout the fungal kingdom. Thus, as antigens, they are less prone to degradation by micro-organisms in soil, thereby rendering them more accessible to extraction and detection. In developing assays for the detection of fungi that lack a mycelial phase or for which the mycelial stage of growth is restricted, spores have been used directly as the immunogen. Screening of hybridoma cell line supernatants against a range of related and unrelated soil-borne organisms is an obvious and necessary prerequisite in the selection of cell lines secreting specific MAbs. Comparative studies are necessary to examine the merits, sensitivity, bias and logistic constraints of immunological and molecular methods. Species specificity can be achieved with MAbs and it is possible to raise separate antibodies to distinguish between spores and mycelium of a single species. This enables quantification of the different components of the fungal population. Greater flexibility can be achieved by selecting two antibodies from different immunoglobulin classes or subclasses that are suited to different assay formats, particularly for immunomagnetic bead assays, where one can be used as the capture and the other as the detector antibody.

Use of Monoclonal Antibodies to Determine Biomass of Cladosporium fulvum in Infected Tomato Leaves

A monoclonal antibody, OX-CH1, was raised against surface washings of Cladosporium herbarum. This antibody recognizes an epitope that is found in various fungi belonging to the genus Cladosporium, including C. fulvum, the causal agent of tomato leaf mold. The epitope is present at comparable levels in two different races of C. fulvum and in transgenic isolates derived from them. The epitope is heat-and protease-resistant but sensitive to oxidation with periodate and it is constitutively expressed in C. fulvum grown in pure culture and on the plant. C. fulvum can be detected in infected tissues at levels starting from around 1 mg fresh weight of fungus per g fresh weight of leaf tissue. Noninfected tomato leaves do not cross-react with OX-CH1. We have developed an enzyme-linked immunosorbent assay (ELISA) for fungal biomass in tomato leaves and compared it with the assay based on measurements of beta-glucuronidase (GUS) activity in tissues infected with a transgenic isolate of C. fulvum race 4 carrying a uidA gene; the two assays give similar results.

Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.