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In JoVE (1)
Other Publications (21)
- Microbiology (Reading, England)
- The International Journal of Eating Disorders
- European Child & Adolescent Psychiatry
- Molecular Microbiology
- Environmental Microbiology
- The Plant Cell
- Phytopathology
- Molecular Microbiology
- Environmental Microbiology
- Nature
- Current Biology : CB
- Nature Protocols
- European Eating Disorders Review : the Journal of the Eating Disorders Association
- Clinical and Vaccine Immunology : CVI
- Methods in Molecular Biology (Clifton, N.J.)
- Clinical and Vaccine Immunology : CVI
- The Plant Cell
- Clinical and Vaccine Immunology : CVI
- Advances in Applied Microbiology
- International Journal of Food Microbiology
- Microbiology (Reading, England)
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Articles by Christopher Thornton in JoVE
JoVE Immunology and Infection
Yanal-flow teknolojisi kullanılarak Hematolojik Malignite Hastalarda invaziv pulmoner aspergillozis Algılama
1Biosciences, University of Exeter, 2BICMS, Queen Mary University of London, 3St. Bartholomew's Hospital and The London NHS Trust
Invaziv pulmoner aspergillozis için hızlı ve hassas noktası bakım testi sunulmaktadır. Bir bağlanan bir monoklonal antikor kullanılarak yanal-akış teknolojisi yararlanır
Other articles by Christopher Thornton on PubMed
Production of a Monoclonal Antibody Specific to the Genus Trichoderma and Closely Related Fungi, and Its Use to Detect Trichoderma Spp. in Naturally Infested Composts
Studies of the interactions between hyperparasitic fungi and their hosts are severely hampered by the absence of methods that allow the unambiguous identification of individual genera in complex environments that contain mixed populations of fungi, such as soil or compost. This study details the development of a monoclonal antibody (MF2) that allows the detection and recovery of Trichoderma spp. in naturally infested composts, and the visualization of hyperparasitic strains of Trichoderma during antagonistic interactions with their hosts. Murine monoclonal antibody MF2, of immunoglobulin class M (IgM), was raised against a protein epitope of a glycoprotein antigen(s) specific for species of the genus Trichoderma and for the closely related fungi Gliocladium viride, Hypomyces chrysospermus, Sphaerostilbella spp. and Hypocrea spp. MF2 did not react with antigens from Gliocladium catenulatum, Gliocladium roseum, Nectria ochroleuca and Clonostachys spp., nor with a range of unrelated soil- and compost-borne fungi. Extracellular production of the MF2 antigen was constitutive. Western-blotting analysis showed that MF2 bound to a ladder of proteins with apparent molecular masses in the range 35-200 kDa. Immunofluorescence studies showed that MF2 bound strongly to the cell walls of hyphae and phialides and the intercalary and terminal chlamydospores of Trichoderma spp., whereas immunogold electron microscopy revealed strong binding of MF2 to the cell walls and septa of hyphae and to the cell walls of phialoconidia. In immunofluorescence studies of dual cultures of Trichoderma and Rhizoctonia solani, only the cell walls of the hyperparasite, which coiled around the host, were stained by MF2. The specificity of MF2 enabled the development of a combined baiting-ELISA technique for the detection of Trichoderma spp. in naturally infested composts. The specificity of this technique was confirmed by phylogenetic analysis based on sequences of the ITS1-5.8S-ITS2 rRNA-encoding regions of the isolates.
The Australian Experience of Day Programs for Patients with Eating Disorders
To describe the authors' experience in establishing and implementing day programs for the treatment of eating disorders in Australia.
The Incorporation of the Stage of Change Model in the Day Hospital Treatment of Patients with Anorexia Nervosa
The development of day hospital programmes for patients with anorexia nervosa has received much interest of late. However, there has often been little attention paid to the unique nature of this disorder. For this reason, we set out to design day hospital treatment programmes to reflect and incorporate an understanding of the ambivalence towards change demonstrated by the majority of such patients. We recognise that, from the sufferers' perspective, anorexia is a functional and ego syntonic illness. It is an illness that on some levels has "adaptive" advantages which the patient does not wish to relinquish. Patients are at different levels of readiness to change aspects of their behaviour. Therefore day hospital treatment programmes must aim to match the type of therapy delivered to the readiness to change of the patient. If there is a 'mismatch' between the stage of change and the goals of treatment, resistance will occur. Using the transtheoretical model of change as a paradigm, we have designed a series of day programmes that aim to meet the needs of patients at different stages of change.
The Glyoxylate Cycle is Required for Temporal Regulation of Virulence by the Plant Pathogenic Fungus Magnaporthe Grisea
We describe the isolation and characterization of ICL1 from the rice blast fungus Magnaporthe grisea, a gene that encodes isocitrate lyase, one of the principal enzymes of the glyoxylate cycle. ICL1 shows elevated expression during development of infection structures and cuticle penetration, and a targeted gene replacement showed that the gene is required for full virulence by M. grisea. In particular, we found that the prepenetration stage of development, before entry into plant tissue, is affected by loss of the glyoxylate cycle. There is a delay in germination, infection-related development and cuticle penetration in Delta icl1 mutants. Recent reports have shown the importance of the glyoxylate cycle in the virulence of the human pathogenic fungus Candida albicans and the bacterial pathogen Mycobacterium tuberculosis. Our results indicate that the glyoxylate cycle is also important in this plant pathogenic fungus, demonstrating the widespread utility of the pathway in microbial pathogenesis.
An Immunological Approach to Quantifying the Saprotrophic Growth Dynamics of Trichoderma Species During Antagonistic Interactions with Rhizoctonia Solani in a Soil-less Mix
Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.
A Fungal Metallothionein is Required for Pathogenicity of Magnaporthe Grisea
The causal agent of rice blast disease, the ascomycete fungus Magnaporthe grisea, infects rice (Oryza sativa) plants by means of specialized infection structures called appressoria, which are formed on the leaf surface and mechanically rupture the cuticle. We have identified a gene, Magnaporthe metallothionein 1 (MMT1), which is highly expressed throughout growth and development by M. grisea and encodes an unusual 22-amino acid metallothionein-like protein containing only six Cys residues. The MMT1-encoded protein shows a very high affinity for zinc and can act as a powerful antioxidant. Targeted gene disruption of MMT1 produced mutants that show accelerated hyphal growth rates and poor sporulation but had no effect on metal tolerance. Mmt1 mutants are incapable of causing plant disease because of an inability to bring about appressorium-mediated cuticle penetration. Mmt1 appears to be distributed in the inner side of the cell wall of the fungus. These findings indicate that Mmt1-like metallothioneins may play a novel role in fungal cell wall biochemistry that is required for fungal virulence.
A One-Step, Immunochromatographic Lateral Flow Device Specific to Rhizoctonia Solani and Certain Related Species, and Its Use to Detect and Quantify R. Solani in Soil
ABSTRACT A murine hybridoma cell line GD2 secreting an immunoglobulin (Ig)M monoclonal antibody (MAb) was produced against surface antigens from an anastomosis group (AG) 4 isolate of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris). Ascites were produced in mice using GD2 hybridoma cells and used to develop a rapid immunochromatographic lateral flow device (LFD) for the detection of antigens from R. solani and certain related Rhizoctonia spp. The LFD was tested for specificity against surface antigens from related and unrelated soil fungi. Antigens from representative isolates of R. solani AGs 1, 2-1, 2-3, 2-t, 3, 4, 5, 6, 7, 8, 9, 10, 11, and BI gave a positive response in LFD tests, as did antigens from Thanatephorus orchidicola, T. praticola, R. fragariae (teleomorph: Ceratorhiza fragariae), Ceratorhiza goodyerae-repentis, Ceratobasidium cornigerum, and binucleate AGE. Antigens from R. solani AGs 2-2, 2-2IIIB, and 2-2IV and from the related fungi R. carotae, R. cerealis (teleomorph: Ceratobasium cereale), R. crocorum (teleomorph: Helicobasidium brebissonii), R. oryzae (teleomorph Waitea circinata), and R. zeae gave negative responses, as did antigens from a range of unrelated fungi and oomycetes including Fusarium, Gliocladium, Trichoderma, Pythium, and Phytophthora spp. The usefulness of the LFD to detect R. solani was demonstrated in soils naturally infested with R. solani AG3. There was close agreement between results of LFD tests and conventional plate enrichment tests employing selective medium. The specificity of the technique was confirmed by polymerase chain reaction PCR using R. solani AG3-specific primers and by analyses based on sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA-encoding regions of unrelated fungi recovered from soil samples. The LFD was used to quantify R. solani AG4 in artificially infested soil samples (chopped potato soil inoculum). Estimates of CFU per gram of soil were derived using a most-probable number technique, which was based on the presence or absence of a detectable signal in the LFD. Estimates of CFU obtained in LFD tests and those obtained in a plate-trapped antigen enzyme-linked immunosorbent assay incorporating MAb GD2 were identical (449 CFU g(-1) of soil).
Four Conserved Intramolecular Disulphide Linkages Are Required for Secretion and Cell Wall Localization of a Hydrophobin During Fungal Morphogenesis
Hydrophobins are morphogenetic proteins produced by fungi during assembly of aerial hyphae, sporulation, mushroom development and pathogenesis. Eight cysteine residues are present in hydrophobins and form intramolecular disulphide bonds. Here, we show that expressing eight cysteine-alanine substitution alleles of the MPG1 hydrophobin gene from Magnaporthe grisea causes severe defects in development of aerial hyphae and spores. Immunolocalization revealed that Mpg1 hydrophobin variants, lacking intact disulphide bonds, retain the capacity to self-assemble, but are not secreted to the cell surface. This provides the first genetic evidence that disulphide bridges in a hydrophobin are dispensable for aggregation, but essential for secretion.
Use of Monoclonal Antibodies to Quantify the Dynamics of Alpha-galactosidase and Endo-1,4-beta-glucanase Production by Trichoderma Hamatum During Saprotrophic Growth and Sporulation in Peat
Trichoderma species are ubiquitous soil and peat-borne saprotrophs that have received enormous scientific interest as biocontrol agents of plant diseases caused by destructive root pathogens. Mechanisms of biocontrol such as antibiosis and hyperparasitism are well documented and the biochemistry and molecular genetics of these processes defined. An aspect of biocontrol that has received little attention is the ability of Trichoderma species to compete for nutrients in their natural environments. Trichoderma species are efficient producers of polysaccharide-degrading enzymes that enable them to colonize organic matter thereby preventing the saprotrophic spread of plant pathogens. This study details the use of monoclonal antibodies (mAbs) to quantify the production of two enzymes implicated in the saprotrophic growth of Trichoderma species in peat. Using mAbs specific to the hemicellulase enzyme alpha-galactosidase (AGL) and the cellulase enzyme endo-1,4-beta-glucanase (EG), the relationship between the saprotrophic growth dynamics of a biocontrol strain of Trichoderma hamatum and the concomitant production of these enzymes in peat-based microcosms was studied. Enzyme activity assays and enzyme protein concentrations derived by enzyme-linked immunosorbent assay (ELISA) established the precision and sensitivity of mAb-based assays in quantifying enzyme production during active growth of the fungus. Trends in enzyme activities and protein concentrations were similar for both enzymes, during a 21-day sampling period in which active growth and sporulation of the fungus in peat was quantified using an independent mAb-based assay. There was a sharp increase in active biomass of T. hamatum 3 days after inoculation of microcosms with phialoconidia. After 3 days there was a rapid decline in active biomass which coincided with sporulation of the fungus. A similar trend was witnessed with EG activities and concentrations. This showed that EG production related directly to active growth of the fungus. The trend was not found, however, with AGL. There was a rapid increase in enzyme activities and protein concentrations on day 3, after which they remained static. The reason for the maintenance of elevated AGL probably resulted from secretion of the enzyme from conidia and chlamydospores. ELISA, immunofluoresence and immunogold electron microscopy studies of these cells showed that the enzyme is localized within the cytoplasm and is secreted extracellularly into the surrounding environment. It is postulated that release of oligosaccharides from polymeric hemicellulose by the constitutive spore-bound enzyme leads to AGL induction and could act as an environmental cue for spore germination.
A P-type ATPase Required for Rice Blast Disease and Induction of Host Resistance
To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver proteins directly into plant cells, where they suppress plant defences and facilitate tissue invasion. How plant pathogenic fungi, which cause many of the world's most serious plant diseases, deliver proteins during plant infection is currently unknown. Here we report the characterization of a P-type ATPase-encoding gene, MgAPT2, in the economically important rice blast pathogen Magnaporthe grisea, which is required for exocytosis during plant infection. Targeted gene replacement showed that MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. DeltaMgapt2 mutants are impaired in the secretion of a range of extracellular enzymes and accumulate abnormal Golgi-like cisternae. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment of rice blast disease involves the use of MgApt2-dependent exocytotic processes that operate during plant infection.
Evolution of Filamentous Plant Pathogens: Gene Exchange Across Eukaryotic Kingdoms
Filamentous fungi and oomycetes are eukaryotic microorganisms that grow by producing networks of thread-like hyphae, which secrete enzymes to break down complex nutrients, such as wood and plant material, and recover the resulting simple sugars and amino acids by osmotrophy. These organisms are extremely similar in both appearance and lifestyle and include some of the most economically important plant pathogens . However, the morphological similarity of fungi and oomycetes is misleading because they represent some of the most distantly related eukaryote evolutionary groupings, and their shared osmotrophic growth habit is interpreted as being the result of convergent evolution . The fungi branch with the animals, whereas the oomycetes branch with photosynthetic algae as part of the Chromalveolata . In this report, we provide strong phylogenetic evidence that multiple horizontal gene transfers (HGT) have occurred from filamentous ascomycete fungi to the distantly related oomycetes. We also present evidence that a subset of the associated gene families was initially the product of prokaryote-to-fungi HGT. The predicted functions of the gene products associated with fungi-to-oomycete HGT suggest that this process has played a significant role in the evolution of the osmotrophic, filamentous lifestyle on two separate branches of the eukaryote tree.
Immunofluorescence Microscopy and Immunogold EM for Investigating Fungal Infection of Plants
Fungi are a diverse group of eukaryotic organisms whose activities are intricately linked to the lives of human beings. Their involvement in plant productivity, as agents of human diseases, as sources of medicines and enzymes and as model experimental organisms has necessitated the development of sensitive and specific techniques for tracking the organisms and their protein products. Techniques employing highly specific monoclonal antibodies have allowed the visualization of fungi in their natural environments and have facilitated the study of their antigens at the subcellular level. Here, we describe three such techniques, immunofluorescence (IF), immuno-enzymatic staining (IES) and immunoelectron microscopy (IEM), that have found widespread applicability in studies of fungal biology, and which can also be adapted for use in the study of other eukaryotic organisms. Results from the IF and IES procedures can be obtained within 4-5 h. Sample preparation for IEM takes approximately 4 days. Gold labeling and visualization of samples can be completed within 4 h.
Group Motivational Enhancement Therapy As an Adjunct to Inpatient Treatment for Eating Disorders: a Preliminary Study
Difficulties in fostering eating disorder (ED) patients' motivations to overcome their illness are widely considered to be a major hurdle in the course of successful treatment. However, no previous study has assessed the use of interventions specifically designed to target poor motivation amongst patients with illnesses that are severe enough to warrant hospitalisation.
Development of an Immunochromatographic Lateral-flow Device for Rapid Serodiagnosis of Invasive Aspergillosis
Aspergillus fumigatus is a cosmopolitan saprotrophic fungus that is second only to Candida species as a cause of invasive fungal infections in immunocompromised humans. Current immunodiagnostic tests for invasive aspergillosis (IA) are based on the detection of circulating galactomannan (GM) in a patient's serum by using a rat monoclonal antibody (MAb), EB-A2, that binds to tetra (1-->5)-beta-D-galactofuranoside, the immunodominant epitope in GM. The potential cross-reactivity of MAb EB-A2 with non-Aspergillus fungi, with contaminating GM in beta-lactam antibiotics and foodstuffs, and with bacterial lipoteichoic acids has prompted efforts to discover non-GM antigens that can act as surrogate markers for the diagnosis of IA. This paper describes the development of a mouse MAb, JF5, that binds to a protein epitope present on an extracellular glycoprotein antigen secreted constitutively during the active growth of A. fumigatus. The MAb was used to develop an immunochromatographic lateral-flow device (LFD) for the rapid (15-min) detection of Aspergillus antigens in human serum. The test is highly specific, reacting with antigens from Aspergillus species but not with antigens from a large number of clinically important fungi, including Candida species, Cryptococcus neoformans, Fusarium solani, Penicillium marneffei, Pseudallescheria boydii, and Rhizopus oryzae. The LFD was able to detect circulating antigen in serum samples from patients suspected of having or shown to have IA on the basis of their clinical symptoms and results from tests for GM and fungal (1-->3)-beta-D-glucan. The ease of use of the LFD provides a diagnostic platform for the routine testing of vulnerable patients who have an elevated risk of IA.
Production of Monoclonal Antibodies to Plant Pathogens
The use of monoclonal antibodies in plant pathology has improved the quality and specificity of detection methods for diseases. Hybridoma technology allows the limitless production of highly specific antibodies which can be used to identify pathogens to the species or even sub-species level.
Tracking the Emerging Human Pathogen Pseudallescheria Boydii by Using Highly Specific Monoclonal Antibodies
Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 kappa-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.
Phylogenomic Analysis Demonstrates a Pattern of Rare and Ancient Horizontal Gene Transfer Between Plants and Fungi
Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries and is an important evolutionary phenomenon in the ancestry of many microbes. The role of HGT in plant evolutionary history is, however, largely unexplored. Here, we compare the genomes of six plant species with those of 159 prokaryotic and eukaryotic species and identify 1689 genes that show the highest similarity to corresponding genes from fungi. We constructed a phylogeny for all 1689 genes identified and all homolog groups available from the rice (Oryza sativa) genome (3177 gene families) and used these to define 14 candidate plant-fungi HGT events. Comprehensive phylogenetic analyses of these 14 data sets, using methods that account for site rate heterogeneity, demonstrated support for nine HGT events, demonstrating an infrequent pattern of HGT between plants and fungi. Five HGTs were fungi-to-plant transfers and four were plant-to-fungi HGTs. None of the fungal-to-plant HGTs involved angiosperm recipients. These results alter the current view of organismal barriers to HGT, suggesting that phagotrophy, the consumption of a whole cell by another, is not necessarily a prerequisite for HGT between eukaryotes. Putative functional annotation of the HGT candidate genes suggests that two fungi-to-plant transfers have added phenotypes important for life in a soil environment. Our study suggests that genetic exchange between plants and fungi is exceedingly rare, particularly among the angiosperms, but has occurred during their evolutionary history and added important metabolic traits to plant lineages.
Comparison of Lateral Flow Technology and Galactomannan and (1->3)-beta-D-glucan Assays for Detection of Invasive Pulmonary Aspergillosis
We compared a lateral flow device to galactomannan and (1-->3)-beta-D-glucan assays to detect invasive aspergillosis in an established guinea pig model of pulmonary disease. The lateral flow device became positive earlier (day 3) than the (1-->3)-beta-D-glucan and galactomannan assays (day 5), with all samples positive by each assay on day 7.
Detection of Invasive Aspergillosis
Invasive aspergillosis (IA) caused by the fungus Aspergillus fumigatus is a frequent and life-threatening complication of chemotherapy and bone marrow transplantation with high rates of mortality and morbidity. Diagnosis of IA is complex and can only be confirmed by identification of the fungus in biopsy samples. Capturing tissue for diagnosis is in itself hazardous, and because of this many patients receive empirical antifungal treatment rather than undergo biopsy. However, the treatment carries with it significant side effects and is prohibitively expensive. Because of this, attempts have been made to develop specific and sensitive diagnostic tests that can be used to track the early onset of infection and permit rational administration of antifungal drugs. Early attempts at nonculture-based diagnosis using human immune serum to detect circulating Aspergillus antigens proved unreliable, and so focus turned to hybridoma technology and the use of monoclonal antibodies (MAbs) to detect signature molecules of infection. Detection of one such signature molecule, galactomannan (and associated galactomannoprotein molecules), forms the basis of the commercial Platelia enzyme immunoassay (EIA), an assay that has found widespread use in IA diagnosis. Nevertheless, concerns surrounding its accuracy mean that alternative strategies to diagnosis have been sought including detection of the fungal cell wall component (1-->3)-beta-d-glucan and polymerase chain reaction (PCR). The poor specificity of "panfungal" (1-->3)-beta-d-glucan tests and current lack of standardization of PCR assays have led to the recent development of next-generation MAb-based assays that detect surrogate markers of infection and that have been incorporated into "point-of-care" diagnostic devices. This chapter examines the development of antibody-antigen, (1-->3)-beta-d-glucan, and nucleic acid-based approaches to IA detection, current concerns surrounding accurate disease diagnosis, and how animal models of infection can be used to inform assay development and validation.
Detection of the Sour-rot Pathogen Geotrichum Candidum in Tomato Fruit and Juice by Using a Highly Specific Monoclonal Antibody-based ELISA
Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes.
Saprotrophic Competitiveness and Biocontrol Fitness of a Genetically Modified Strain of the Plant-growth-promoting Fungus Trichoderma Hamatum GD12
Trichoderma species are ubiquitous soil fungi that hold enormous potential for the development of credible alternatives to agrochemicals and synthetic fertilizers in sustainable crop production. In this paper, we show that substantial improvements in plant productivity can be met by genetic modification of a plant-growth-promoting and biocontrol strain of Trichoderma hamatum, but that these improvements are obtained in the absence of disease pressure only. Using a quantitative monoclonal antibody-based ELISA, we show that an N-acetyl-β-d-glucosaminidase-deficient mutant of T. hamatum, generated by insertional mutagenesis of the corresponding gene, has impaired saprotrophic competitiveness during antagonistic interactions with Rhizoctonia solani in soil. Furthermore, its fitness as a biocontrol agent of the pre-emergence damping-off pathogen Sclerotinia sclerotiorum is significantly reduced, and its ability to promote plant growth is constrained by the presence of both pathogens. This work shows that while gains in T. hamatum-mediated plant-growth-promotion can be met through genetic manipulation of a single beneficial trait, such a modification has negative impacts on other aspects of its biology and ecology that contribute to its success as a saprotrophic competitor and antagonist of soil-borne pathogens. The work has important implications for fungal morphogenesis, demonstrating a clear link between hyphal architecture and secretory potential. Furthermore, it highlights the need for a holistic approach to the development of genetically modified Trichoderma strains for use as crop stimulants and biocontrol agents in plant agriculture.
