R.J. O'Connell

Cytology and physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum

Abstract The progress of infection by Colletotrichum lindemuthianum was examined in susceptible and resistant French bean hypocotyls producing spreading lesions or single hypersensitive cells, respectively. In susceptible tissue, intracellular infection vesicles formed in epidermal cells, which remained alive. Intracellular primary hyphae developed from the vesicles and colonized further host cells. A matrix layer separated the hyphal wall from the invaginated host plasmalemma. After a period of biotrophy lasting less than 24 h, the cytoplasm of infected cells gradually degenerated. This was associated with loss of the ability of cells to plasmolyse and to exclude tannic acid, here used as a permeability tracer with plant tissue for the first time. Loss of the ability of the tonoplast to contract and for neutral red to accumulate in the vacuole occurred later, and was considered to indicate cell death. In cultivars containing the pigment malvidin-3,5-diglucoside, loss of colour coincided with tonoplast rupture. During the development of the primary mycelium, the sequence of a brief biotrophy phase followed by gradual degeneration and death was repeated as each host cell became infected. Thus, despite the absence of tissue browning, only recently colonized cells at the edge of the infection were alive. As lesions appeared, narrower secondary hyphae grew within host cell walls. Death of host protoplasts and wall dissolution then occurred in advance of secondary hyphae. In resistant tissue, infection vesicles were not formed, and the fungus was restricted in single hypersensitive epidermal cells. Most hyphae appeared dead, but some had normal ultrastructure. These findings are discussed in relation to race specificity and the importance of biotrophy to successful pathogenesis by C. lindemuthianum.

Infection process and identity of the hemibiotrophic anthracnose fungus (Colletotrichum destructivum) from cowpea (Vigna unguiculata)

The infection process of a hemibiotrophic Colletotrichum species causing anthracnose disease in cowpea (Vigna unguiculata) was studied by light and electron microscopy. During the biotrophic phase, the fungus produced unusual, large, multilobed, multiseptate infection vesicles with elongated neck regions, which remained confined within the initially-infected epidermal cell. Biotrophy lasted for the first 72 h of the host-pathogen interaction. The necrotrophic phase was characterized by the rapid development of invasive secondary hyphae which radiated from the multilobed vesicles into the surrounding tissues, producing water-soaked lesions on the surface of infected organs. Eventually, acervuli erupted in abundance on the surface of colonized tissues 120 h after inoculation. Each acervulus possessed, characteristically, one septate melanized seta. Multilobed vesicles were successfully isolated from homogenates of infected leaves by isopycnic centrifugation and were shown to lack cross-reactivity to monoclonal antibodies raised against infection structures of C. lindemuthianum. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed very close similarities (97–99%) between the cowpea isolate and three isolates of C. destructivum obtained from lucerne (Medicago sativa). The results presented here, in addition to other morphological and growth attributes, suggest that the hemibiotrophic cowpea anthracnose pathogen must be considered as a form of C. destructivum.

Monoclonal antibodies which show restricted binding to four Colletotrichum species: C. lindemuthianum, C. malvarum, C. orbiculare and C. trifolii☆

Monoclonal antibodies (MAbs) were raised to Colletotrichum lindemuthianum race γ germlings. When screened against eleven other Colletotrichum species using an ELISA, two of these MAbs, UB20 and UB22, showed restricted cross-reaction with three other species, viz. C. malvarum, C. orbiculare and C. trifolii. It was also observed that UB20 and UB22 bound only to isolates of C. lindemuthianum from bean and not cowpea. These findings suggest that the four Colletotrichum species recognized by the two MAbs are very closely related, confirming previous evidence. The MAbs did not bind to species of Fusarium or Phoma. UB20 and UB22 bound to carbohydrate epitopes on two distinct sets of glycoproteins. The glycoproteins recognized by the two MAbs were present on the surface of different infection structures in varying amounts: UB20 antigens were predominantly located on conidia, whereas UB22 antigens appeared to be most abundant in fibrillar material around germ-tubes. Both MAbs labelled the wall and matrix around intracellular infection hyphae. Potential applications of the MAbs for the identification of the above pathogens are discussed.

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

SummaryThe ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 μm long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, α-D-mannose and α-D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (Mr 133,000 and 146,000). Reductions in the Mr of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.

Ultrastructure and composition of the cell surfaces of infection structures formed by the fungal plant pathogen Colletotrichum lindemuthianum

A variety of microscopical techniques and molecular probes have been used to study the ultrastructure and composition of the cell surfaces of the conidia (i.e. spores) and infection structures produced by the hemibiotrophic fungal plant pathogen Colletotrichum lindemuthianum. The fungal conidium germinates to produce a germ‐tube, the tip of which swells to produce a domed, melanized appressorium which adheres firmly to the plant surface. Penetration of the cuticle and cell wall is followed by the development of a biotrophic intracellular hypha, which is surrounded by an invagination of the host plasma membrane. Freeze‐substitution of C. lindemuthianum germlings showed that conidia are coated with a dense layer of fibrillar material. This ‘spore coat’ contains irregularly shaped pores, giving it a reticular appearance. Negative staining of germlings revealed the presence of numerous long, flexuous fibres or fimbriae, protruding from the surfaces of germ‐tubes and appressoria. Colloidal gold was used to visualize fungal extracellular proteins. The colloidal gold stained a fibrillar sheath around germ‐tubes, whereas appressoria were surrounded by a halo, comprising an inner unstained region and a stained perimeter. The carbohydrate composition of the cell surfaces of the conidia and infection structures was studied by labelling cells with rhodamine‐ and fluorescein‐conjugated lectins. The results showed that the extracellular matrices of germ‐tubes and appressoria are very similar in composition, but differ from those of conidia and intracellular hyphae. Monoclonal antibodies have been prepared to germlings and infection structures of C. lindemuthianum and their use has provided further evidence that the extracellular matrices around germ‐tubes and appressoria have several glycoproteins in common. The results also show that the cell surface of C. lindemuthianum becomes specialized during biotrophic development inside host cells.

Infection process and host specificity of a Colletotrichum species causing anthracnose disease of cowpea, Vigna unguiculata.

The morphology, pathogenicity and host specificity of a strain of C. lindemuthianum (I57), causing anthracnose on cowpea were studied. Strain I57 exhibited a novel hemibiotrophic infection process which resulted in production of water-soaked lesions in all tissues of cowpea seedlings. Biotrophic infection hyphae formed in viable epidermal cells: the hyphae were large and became highly branched, but remained within the initially-infected epidermal cells. After 20–30 h, the infected epidermal cells died and soon afterwards the surrounding uninfected cells also died. When the initially-infected epidermal cells had been extensively colonized, thinner sparsely branched hyphae grew into and through the surrounding tissues, producing lesions with large numbers of acervuli on their surfaces. This type of infection process has not previously been described for a species of Colletotrichum . When inoculated on to hypocotyls of bean ( Phaseolus vulgaris ) and several other legumes, I57 caused rapid hypersensitive death of single epidermal cells. A comparison of strain I57 with isolates of C. lindemuthianum that attack bean, but not cowpea indicated that their culture morphologies, mycelial growth rates, modes of conidial germination, infection processes and host specificities were different. These results suggest that the identification of strain I57 as a form of C. lindemuthianum should be re-examined.

Surface characteristics of necrotrophic secondary hyphae produced by the bean anthracnose fungus, Colletotrichum lindemuthianum

During infection of bean (Phaseolus vulgaris), the hemibiotrophic anthracnose pathogen, Colletotrichum lindemuthianum, initially produces biotrophic primary hyphae that are large-diameter and entirely intracellular, followed by necrotrophic secondary hyphae that are narrower and either intercellular or intracellular. In the present study, transmission electron microscopy of infected tissues prepared by high-pressure freezing and freeze-substitution showed that secondary hyphae have much thinner cell walls (25–40 nm) than primary hyphae (100–130 nm) and are not surrounded by an extracellular matrix. Immunofluorescence labelling with a panel of monoclonal antibodies showed that glycoproteins which are present on conidia, germ-tubes, appressoria, primary hyphae and mycelium grown in vitro are absent from the surface of secondary hyphae. Chitin, detected with the lectin wheat germ agglutinin, was the only surface component shared by secondary hyphae and the other fungal cell types. The results suggest that the fungal cell surface becomes modified during necrotrophic growth, with none of the glycoproteins associated with earlier stages of the infection process being produced.

Sensitive staining of fungal extracellular matrices using colloidal gold

A method is described for visualizing fungal extracellular matrices (ECMs), based on the binding of non-conjugated colloidal gold particles. Staining is permanent and is viewed either with epi-polarization microscopy or with bright field microscopy after silver enhancement with a physical developer. Colloidal gold bound to cell surfaces through ionic interactions with the free amino groups of basic proteins. Sensitivity of the stain was high: five other protein stains (Orange G, Light Green SF, Alkaline Fast Green FCF, fluorescein isothiocyanate and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide), failed to label extracellular material that was stained by colloidal gold. Colloidal gold was used to demonstrate ECMs around germlings of four plant pathogenic fungi attached to artificial and host surfaces. Large circular films of protein were detected around conidia of Colletotrichum gloeosporioides and Erysiphe graminis and around appressoria of Magnaporthe grisea. Colloidal gold also revealed fibrillar protein sheaths enveloping germ-tubes of C. gloeosporioides and Botrytis cinerea.

Identification and localisation of glycoproteins in the extracellular matrices around germ-tubes and appressoria of Colletotrichum species

A monoclonal antibody (MAb), UB31, is described that binds to the extracellular matrix (ECM) surrounding germtubes and appressoria, but not conidia, of the bean anthrachose fungus, Colletotrichum lindemuthianum. Comparative localisation studies with MAb UB26, which has the same cell type specificity, suggest that the ECM is heterogeneous in composition. Immunofluorescence showed that UB31 labelled appressoria more intensely than germ-tubes, whereas UB26 labelled these structures to a similar extent. Immunofluorescence and TEM-immunogold labelling showed that UB31 antigens were located close to the appressorial wall, while UB26 antigens extended further away from the wall. MAb UB31 bound to the ECMs of all six Colletotrichum species tested. Western blotting and ELISA indicated that the antibody recognises a high M r glycoprotein (> 200000) that may be linked to melanin. The glycoprotein recognised by UB31 was not removed from substrata by ultrasonication, suggesting it may contribute to germling adhesion.

A plasma membrane-associated protein is a marker for differentiation and polarisation ofColletotrichum lindemuthianum appressoria

SummaryThe appressorium formed by the facultative biotrophic fungusColletotrichum lindemuthianum on bean tissues is a specialised cell involved in penetration of the host cuticle and epidermal cell wall, leading to the formation of intracellular hyphae. A monoclonal antibody designated UB27, raised against infection structures isolated from infected leaves ofPhaseolus vulgaris, bound specifically to appressoria, as shown by immunofluorescence, EM-immunogold and Western blotting. It did not bind to other fungal or plant structures. Immunogold labelling of appressoria formed on bean hypocotyls showed that UB27 bound to the appressorial plasma membrane and a layer of cytoplasm just beneath this membrane. Labelling stopped abruptly at the point at which the appressorial wall contacted the plant cuticle, leaving a region that included the penetration pore and appressorial cone unlabelled. Labelling in appressoria formed on polycarbonate membranes was similar, except that the diameter of the unlabelled region was smaller. UB27 recognised a 48–50 kDa protein. The mobility of this protein was unaffected by peptide-N-glycosidase treatment, but trifluoromethane sulphonic acid treatment resulted in a reduction of Mr of approx. 16000. This suggests that the protein is glycosylated, possibly withO-linked carbohydrate side chains. After solubilisation and phase-separation of appressorial proteins in Triton X-114, the protein recognised by UB27 partitioned primarily into the detergent phase, suggesting that it is an integral membrane protein. A proportion of the protein remained unsolubilised, suggesting that there are interactions between the protein and cytoskeletal and/or cell wall components. Overall, the results show that the plasma membrane of appressoria ofC. lindemuthianum is differentiated into two distinct domains and the distribution of the protein identified by UB27 provides evidence for polarisation of appressoria.