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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.

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A genus-specific monoclonal antibody, NG-CF10, raised in a previous study to the fungal pathogen Nectria galligena, was found to recognize the aquatic hyphomycete Heliscus lugdunensis (anamorph) and its teleomorph Nectria lugdunensis. Using this MAb in a plate trapped antigen- ELISA we could detect and determine the biomass of Heliscus lugdunensis in mixed assemblages in both naturally occurring and artificially inoculated leaves and roots of Alnus glutinosa trees. Initial studies indicate that the biomass associated with naturally occurring leaf material is significantly lower than that recorded with laboratory inoculated leaves, suggesting that biomass production is limited in the natural environment. Significantly lower biomass was associated with roots when compared with leaf material, which supports the proposition that rather than a major substrate for the growth of aquatic hyphomycetes, roots act as a refugium for fungal growth.

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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 of Mycosphaerella brassicicola and conidia of Botrytis cinerea by an enzyme-linked immunosorbent assay (ELISA) under controlled environmental conditions. For ascospores of M. brassicicola correlation coefficients (r(2)) 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 of Brassica 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.

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Botrytis cinerea was detected and quantified in pear stems from six orchards in the Pacific Northwest, and changes in fungal biomass in the stems after 6 and 8 months of cold storage in regular (air) atmosphere were studied. The fungus was detected by plating stem halves on selective medium and by enzyme-linked immunosorbent assay (ELISA) using the Botrytis-specific monoclonal antibody BC-12.CA4. Both methods demonstrated that the incidence of B. cinerea increased from 6 to 8 months, but ELISA was more sensitive than standard isolation. Quantitative ELISAs on stems indicated that over 200 µg of B. cinerea biomass per gram of stem tissue was present in the stems of visibly rotted fruits, but usually less than 35 µg/g was present in stems from fruits without visible gray mold. Aureobasidium pullulans, Penicillium spp., Alternaria spp., and Cladosporium spp. were commonly isolated from stem tissue. A. pullulans was present in 86% of the stems from which B. cinerea was detected. Use of the monoclonal antibody BC-12.CA4 could help in determining the infection path of B. cinerea in pear stems and detection of latent infections, enabling the timing and method of control of stem end rot to be optimized.

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We aim to develop a rapid, accurate and sensitive diagnostic assay with which to detect the surface antigens of fungi thought to be involved in allergic fungal rhinosinusitis (AFRS), by assessing the usefulness of immunofluorescence microscopy (IMF) and enzyme linked immuno-absorbent assays (ELISA). The age, sex, clinical symptoms and signs, imaging (CT and/or MRI), microbiological subculture data, sinus contents, blood eosinophilia, aspergillosis precipitins, radioallergoabsorbent technology (RAST) for fungal allergens and histopathology were performed on individuals undergoing endoscopic sinus surgery for suspected AFRS. Thirteen patients were examined, and five monoclonal antibodies raised to the surface washings of various fungi were found to recognize and differentiate between fungal species implicated in sinus disease, i.e. Aspergillus niger, Alternaria alternata, Cochliobolus lunata, Penicillium expansum and Cladosporium species. The IMF microscopy proved to be a useful assay to distinguish visually between the cultured fungi, but was less useful for visualization of fungi in the patient samples. However, ELISA assays with 5 monoclonal antibodies gave clear and unambiguous data as to the presence of certain fungi within the patient samples. There is good correlation between the ELISA data and the pathology findings. This preliminary study suggests that both IMF and ELISA techniques may offer an important advance in this area.

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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.

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In studies with a laboratory isolate of the fungal pathogen Stagonospora (Septoria) nodorum three different isolates of bacteria were closely associated with the fungus. Bacteria were also closely associated with fresh isolates of S. nodorum obtained from artificially and naturally infected field material. Although a range of bacteria was isolated, only one type of bacterium was found to be associated with each isolate of S. nodorum. In co-inoculation studies with pycnidiospores of the fungus on detached leaves, some of the bacterial isolates significantly increased the pathogenicity of the fungus, particularly Xanthomonas maltophilia, Sphingobacterium multivorum, Enterobacter agglomerans and Erwinia amylovora. Evidence is presented indicating that one of the ways that the 'helper bacteria' may assist in the establishment of infections is by the production of lipases that were not detected in germinating fungal spores.

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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.

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The procedure currently used to diagnose infection in otitis externa has several limitations: it is slow to culture organisms on growth media, fungal infections are often missed, and extensive laboratory facilities and mycological expertise are required. A rapid, accurate and sensitive assay would greatly improve patient care by initiating appropriate antifungal treatment at the onset of disease. We report the development of a rapid detection assay for otomycosis using fungal-specific monoclonal antibodies to detect fungi in ear swabs by immunofluorescence microscopy. This assay could form the basis of a detection assay for fungal infections of the head and neck.

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ABSTRACT A murine monoclonal antibody (MAb) of immunoglobulin class M (IgM) was raised against surface antigens from Gaeumannomyces graminis var. tritici and, by enzyme-linked immunosorbent assay, recognized isolates of G. graminis var. tritici, G. graminis var. avenae and G. graminis var. graminis. Characterization of the antigen by heat and protease treatments showed that the epitope recognized by the MAb was a protein. Antigen production was detected only in live mycelia. Immunofluorescence studies showed that the antigen was associated with both the broad melanized macrohyphae and hyaline mycelia of G. graminis var. tritici. Secretion of antigen into an aqueous minimal medium was promoted only by exposure of live mycelia to certain phenolic substrates, including monophenols ortho-, para-, and meta-cresol; 3,4,5-trihydroxybenzoic acid (gallic acid); and phenolic amino acid L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA). Antigen secretion was not promoted by 3-(4-hydroxyphenyl) alanine (L-tyrosine). The MAb reacted strongly with purified enzyme laccase (polyphenol oxidase, EC 1.10.3.2) but did not recognize purified tyrosinase (monophenol oxidase, EC 1.14.18.1). Moreover, chemicals that bind to copper and inhibit copper-containing enzymes such as laccase completely inhibited antigen secretion in response to L-DOPA. The MAb was tested for specificity against a wide range of fungi, common yeast species, and gram positive and negative bacteria. It did not recognize antigens from a broad range of unrelated fungi, including Gliocladium roseum, Fusarium sp., Phoma exigua, Phialophora fastigiata, Penicillium crustosum, Pythium ultimum, Rhizopus stolonifer, Rhizoctonia carotae, R. oryzae, R. tuliparum, and Trichoderma viride, nor did it recognize surface antigens from yeasts or bacteria. The MAb cross-reacted with antigens from Botrytis spp., Chaetomium globosum, R. cerealis, and R. solani. However, secretion of antigen by R. solani and R. cerealis was not promoted by L-DOPA, and secretion by C. globosum in response to the phenolic amino acid was significantly less compared to G. graminis var. tritici.

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Quantitative ELISAs have been developed to determine the biomass of two aquatic hyphomycetes in natural mixed assemblages. These assays employ a species specific rat monoclonal antibody (MAb) raised to Alatospora acuminata and a genus-specific murine MAb raised to Tetracladium marchalianum. The respective antigens are produced constitutively and their production is not affected by a range- of culture conditions. The MAbs can also be used to study the spatial distribution of these fungi using Immunofluorescence. Both antibodies recognize carbohydrate epitopes and belong to the immunoglobulin class IgM. The potential applications of these immunoassays are discussed.

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Monoclonal antibodies (MAbs) to the aquatic hyphomycete Anguillospora longissima were raised in mice by using a coimmunization program. A cell line was raised that produced a MAb of the immunoglobulin M class that was specific for A. longissima both in an enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence but that did not recognize other members of the aquatic hyphomycetes. This MAb (AL-HH8c) was used to develop a quantitative ELISA in vitro. The antigen recognized by AL-HH8c is produced throughout the mycelium, irrespective of mycelial age and culture conditions. By using this MAb, mycelium of A. longissima colonizing leaf material can be detected by ELISA, immunofluorescence, and immunoenzymatic staining methods.

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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.

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Surface washings were prepared from cultures of fungi grown on agar slants and the wash components were fractionated by gel-filtration HPLC. A range of low-Mr glycoproteins (less than 10,000) were detected in all the washes, and in some species several high-Mr glycoprotein components (greater than 150,000) were also present. The HPLC analyses indicated species-specific differences in the profiles. Regions of specific antigenicity within profiles were detected by monoclonal antibodies.

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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.

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