Determination of Coniothyrium minitans conidial and germling lectin avidity by flow cytometry and digital microscopy

Mycol. Res. 103 (12) : 1533–1539 (1999)

1533

Printed in the United Kingdom

Determination of Coniothyrium minitans conidial and germling lectin avidity by flow cytometry and digital microscopy

S. N. S M I T H1, R. A. A R M S T R O NG1, M. B A R K ER1, R. A. B I R D1, R. C H O H AN1, N. A. H A R T E L L1 A N D J. M. W H I P P S2 " Department of Pharmaceutical and Biological Sciences, Aston University, Birmingham, B4 7ET, U.K. # Plant Pathology and Microbiology Department, Horticultural Research International, Wellesbourne, Warwick, CV35 9EF, U.K.

The avidity of conidia and 48-h-old germlings of Coniothyrium minitans for FITC-conjugated lectins was characterised by flow cytometry and digital microscopy. Six isolates of C. minitans representing three morphological types were compared. Binding of Con A, SBA and WGA by conidial populations varied markedly in extent and pattern between isolates, however, with increasing culture age, conidia from all isolates demonstrated a significant reduction in lectin avidity. Germling isolates bound significantly different amounts of lectins and lectin binding differed significantly with locality. Spore walls of all germlings from all isolates bound more Con A compared with hyphal apices and mature hyphal walls. In contrast, hyphal apices of the majority of germling isolates, readily bound SBA and mature hyphal walls of germling isolates bound more WGA than other regions of the germlings. Such differential lectin binding by conidia and germlings may influence their specific surface interactions and adherence characteristics.

Lectins have been widely recognized as major contributors to microbial pathogenicity through their recognition of, and adhesion to, carbohydrate receptors (Doyle, 1994). Specific forces of microbial adhesion are mediated by such entities, manifest as defined microbial surface protein-host carbohydrate interactions, which in turn can give rise to irreversible adhesion (Marshall, 1991). The occurrence and nature of lectins or adhesins amongst fungal pathogens and in diverse aspects of fungal pathology has also received considerable attention (Nicholson & Epstein, 1991 ; Inbar & Chet, 1997). Adhesion of fungal vertebrate pathogens such as Candida albicans and Cryptococcus neoformans to mammalian cells may be mediated by a highly specific and complex multireceptor system, orchestrated by lectins and lectin like entities such as glycosphingolipids and lactosylaceramides. These common moieties, present on the surface of many mammalian cell lines, in association with specific C. albicans cell surface effector molecules of mannoprotein and chitin origin, potentially support the adhesion of C. albicans to diverse mammalian tissues (Korting & Ollert, 1994). Lectin and adhesin mediated attachment or recognition is not restricted to vertebrate fungal interactions alone but has also been frequently demonstrated to occur in other fungal host associations. In contrast to Candida studies, which have concentrated on unicellular entities, investigation of lectin involvement in fungal plant host interactions has demonstrated differential lectin, adhesin and receptor distribution amongst many morphological structures associated with infection, colonisation and adhesion. O’Connell et al. (1996) demonstrated that cell surfaces of the plant pathogen Colletotrichum lindemuthianum bound a panel of fluorescent lectins to differing

degrees, with germ tubes and appresoria showing markedly greater affinity for the panel lectins compared with conidia and particularly intracellular hyphae. Suppression of surface cell wall component expression during rust fungal development has been noted by Freytag & Mendgen (1991). Wheat germ agglutinin (WGA), which binds chitin, had greatest affinity for appressoria and germ tubes, in contrast to infection hyphae, intraepidermal vesicles and substomatal vesicles, suggesting that chitin was masked in the latter structures thereby reducing host defence responses. Attention has been drawn by Inbar & Chet (1997) to the involvement of lectins in recognition, parasitic responses and possible mediation of signal transduction amongst mycoparasites with biocontrol potential. Furthermore, elucidation of the molecular biology and mechanisms of lectin interactions within mycoparasitism may lead to more efficacious biocontrol agents. This paper characterizes fluorescent lectin adherence to conidia and germlings of Coniothyrium minitans W. A. Campb. a sclerotial mycoparasite, which has been shown to control the plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary in a range of glasshouse and field crops (Whipps & Gerlagh, 1992 ; Budge et al., 1995 ; Evenhuis et al., 1995 ; Luth, 1995 ; McQuilken & Whipps, 1995 ; McQuilken et al., 1995). Marked lectin accumulation has been demonstrated to occur within sclerotia of potential C. minitans hosts and, although ascribed as storage proteins, Kellens, Goldstein & Peumans (1992) also postulate that lectins may fulfil other functions for members of the Sclerotinaceae. Studies of lectin receptors on the surfaces of those mycoparasite entities which establish host contact, such as spores and germ tubes, should foster greater understanding of the role of lectin like entities

Conidial and germling lectin avidity in mycoparasitism and fungal adhesion. These studies may also reveal the dynamic nature of such entities as well as refine investigative procedures employed to elucidate lectin and adhesin involvement in mycoparasitism. Fluorescein isothiocynate conjugated lectins have long shown potential in studies designed to evaluate the surface lectin receptors of fungal hyphae and associated infection structures (Mendgen, Lange & Bretschneider, 1985 ; Pendland & Boucias, 1986). The use of fluorescent lectins has been further refined by Bourett, Cyzmmek & Howard (1998), who demonstrated the avidity of fungal intracellular structures and organelles for FITC-Con A. Such studies did not, however, fulfil their full potential due to restricted quantification procedures and limited image analysis. In this study we have employed both flow cytometry and digital microscopy to determine fluorescent lectin binding by conidia and germling surfaces of C. minitans, approaches which support the quantitative measurement of lectin binding by conidial populations and the quantitative determination of spatial lectin distribution along hyphae. MATERIALS AND METHODS

1534 samples for 30 min (as above). The conidial pellets were twice resuspended in PBS and pelleted by centrifugation and then conidia were finally resuspended in 300 µl filtered 1 % v\v formaldehyde\PBS and stored at 4m in the dark until determination of fluorescence. Conidia from 18-d-old cultures of A2 960\1 and B1300 were also incubated with respective inhibitory haptens, to determine whether non-specific adhesion of FITC lectins was a major contributor to fluorescence determination. FITC-conjugated Con A, SBA and WGA were preincubated for 15 min at 4m in 100 m (supplemented PBS) methyl-α- mannopyranoside, N-acetyl- galactosamine and N-acetyl- glucosamine (Sigma, U.S.A.) respectively before addition of conidial suspensions. Conidial samples were subsequently treated in the manner outlined above. Fluorescence was determined with a Becton Dickinson FACS 440 (fluorescence activated cell sorter) using an argon laser (200 mW), excitation wavelength 488 nm, emitted light detector 530 nm (p15 nm), adjusted to a fixed channel using standard Brite Beads (Coulter, U.S.A.) prior to determining fluorescence. Conidia were briefly vortexed before introduction to filtered PBS sheath fluid and fluorescence measured from 10 000 conidia.

Isolates and culture methods

Determination of fluorescent lectin binding to germlings

Five dark isolates of Coniothyrium minitans as described by Smith et al. (1998), were used, together with an additional isolate G8, colony type 1 (isabelline – moderate yellowish brown on top and below) (Sandys-Winsch et al., 1993). These isolates were maintained on Oxoid potato dextrose agar from which sporulating cultures were prepared in the manner outlined by Smith et al. (1998).

Conidia from 18-d-old cultures of each isolate were harvested separately from three replicate plates as outlined above and their concentration adjusted to 1i10( conidia ml−" conidia ml−" in PBS. Aliquots of 20 µl from each spore suspension were dispensed in to wells of separate multispot microscope slides (Hendley, U.K.) and air dried under filtered air. Each slide was subsequently flooded with 20 ml potato dextrose broth (Difco Laboratories, U.S.A.) and incubated at 20m for 48 h. Slides with adherent germlings were rinsed with sterile distilled water, air dried and then 7n5 µl acetone was dispensed in to each well. After the acetone had evaporated the slides were finally rinsed with distilled water and allowed to dry. Each well was flooded with 40 µl of FITC-conjugated lectin (200 µg lectin ml−" in supplemented PBS) and the slides then incubated for 30 min at 4m in the dark. Slides were rinsed with distilled water prior to immediate fluorescence determination. Germlings derived from 18-d cultures in the manner outlined above were also incubated with inhibitory haptens. FITC lectins were preincubated with their respective inhibitor (100 m) for 15 min before addition to wells, after which slides were processed as above. Greyscale 10 bit fluorescent images were collected with a cooled CCD camera (Hamamatsu) attached to an Olympus BX50WI microscope fitted with i40 water immersion objective (N.A. l 0n8). A monochromator (TILL) was used to provide excitation light at a wavelength of 488 nm and emitted light of wavelengths above 505 nm were sent to the camera. The image exposure periods for Con A, SBA and WGA were 200, 1250 and 500 ms respectively. The image exposure times varied to ensure that the full image bit depth was used and images were not saturated. Fluorescence levels from arbitrarily defined regions of similar area (spore, mid-hypha and tip) of five germlings per replicate were analysed with Openlab image analysis software (Improvision).

Determination of fluorescent lectin binding to conidia Conidia from colonies of varying age were harvested by addition of 3 ml pH 7n3 filtered (0n2 µm) phosphate buffered saline (PBS, Oxoid, U.K.) aliquots to three replicate agar plates of each isolate, followed by gentle scraping of each colony surface with a sterile bent rod. Conidial suspensions from each agar plate were then transferred to separate acid-washed, silanized glass universal bottles and stored at 5 mC for a maximum of 45 min until diluted to a concentration of 1i10( conidia ml−" in PBS. Aliquots of 1n5 ml were transferred to individual Eppendorf tubes and centrifuged (10 000 g) for 30 min at 4m. The supernatants were discarded, and the conidial pellets were suspended in fresh PBS, centrifuged and finally resuspended in filtered PBS supplemented with 100 µ CaCl , MnCl and MgCl . Conidial suspensions of # # # 100 µl, were gently mixed with 200 µl fluorescein isothiocyanate (FITC) conjugated lectin (200 µg lectin ml−" in supplemented PBS). The FITC-conjugated lectins employed were concanavalin A (Con A), 3n6 moles FITC mole−" lectin ; soybean agglutinin (SBA), 2 moles FITC mole−" lectin and wheat germ agglutinin (WGA), 2 moles FITC mole−" lectin (Sigma, U.S.A.). The specificities and nature of lectins employed have been outlined by Van Damme et al. (1998). Samples were incubated at 4m in the dark for 30 min, then residual FITC labelled lectin was removed following centrifugation of

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Statistical analysis

heterogeneity of conidial populations is also apparent. Irrespective of strain and sampling period the binding of fluorescent lectins to members of conidial populations shows characteristic patterns. The distributions of Con A and SBA fluorescence are biased towards greater numbers of conidia in channels which represent higher fluorescent determinations. Such a distribution, although manifest amongst WGA labelled conidia, is accentuated in the form of a distinct shoulder on the longer tail of the distribution, representing a sub-population of conidia which potentially demonstrate greater avidity for WGA. Figs 2–4 demonstrate the extent of lectin binding to conidia of differing strains and ages. Molecular weights and conjugation of fluorescein isocyanate varies between the lectins employed precluding quantitative statistical comparison, in particular Con A is conjugated with approximately one and half times more fluorescent moiety than other lectins employed, yet potentially three times fewer Con A moieties exist in reaction mixtures compared to WGA entities. Conidia from all strains under investigation appeared, however, to show marked avidity for Con A considering the log scales

The data were analysed using a two factor, split-ANOVA (SuperANOVA software, Abacus Concepts Inc., Berkley, U.S.A.) with subsequent comparisons between group means using standard errors and confidence intervals appropriate to the split plot design (Snedecor & Cochran, 1980). (i), differences between conidial strain and sampling times were analysed for each fluorescent lectin. (ii), the influence of an inhibitory hapten on fluorescent lectin binding to conidia derived from 18-d-old cultures of A2 960\1 and B1300 was analysed. (iii), differences between germling strain, location and specific affinity were analysed for each fluorescent lectin. RESULTS Conidial fluorescent lectin avidity Fluorescent lectin binding to conidia as determined by flow cytometry was ascertained from similar profiles to those represented in Fig. 1, from which the homogeneity or 170

170

127 Channels Log Con A fluorescence

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Fig. 1. Representative flow cytometry distributions of FITC-conjugated Con A, SBA and WGA binding by conidia from 18-d cultures of A2 960\1. 60

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Figs 2–4. Effect of culture age on conidial binding of FITC-conjugated Con A, SBA and WGA. A l 95 % confidence limits for differences between times within an isolate and B l 95 % confidence limits between two isolates at the same or different times, (i, A2 960\1 ; $, B1300 ; , CH1 ; #, CH2 ; >, G8 ; , IMI 134523 ; all readings adjusted for background\autofluorescence).

Conidial and germling lectin avidity

Isolate A2 960 Con A SBA WGA Isolate B1300 Con A SBA WGA

Lectinjinhibitory hapten

174n5 85n5 60n2

57n0 37n6 27n0

178n4 86n8 74n3

23n3 11n0 19n6

... (95 % confidence level) between lectins and appropriate inhibitory haptens : Con A : methyl-α- mannopyranoside, 4n3 ; SBA : N-acetyl- galactosamine, 10n5 ; WGA : N-acetyl- glucosamine, 8n8.

employed, followed by SBA and WGA respectively. Some marked differences between strains were also apparent (P 0n001), particularly young conidia derived from strain G8 which bind less Con A and WGA than other strains. Young conidia showed greater avidity for fluorescent lectins tested than those derived from older cultures as the capacity to bind fluorescent lectins fell significantly with culture age (Con A P 0n001, SBA P 0n001, WGA P 0n001). Analysis of such results also demonstrated a significant interaction factor (P 0n001 all lectins\strains) indicating that not all strains showed similar reduction in lectin binding with increasing culture age. Although the amount of conidial fluorescence observed after lectin inhibitory hapten incubation significantly differed between strains (P 0n001), Table 1 demonstrates that preincubation of test lectins with appropriate inhibitory haptens significantly reduced lectin binding by conidia from representative strains (P 0n001 all lectins) suggesting that the majority of fluorescence observed in Figs 2–4 is due to specific lectin receptor binding.

Con A fluorescence

Lectin

Spore Middle Tip

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IMI CH2 134523

CH1 B1300 A2 960 G8

A2 960 B1300 + methyl-a-D mannopyranoside

Fig. 5

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Table 1. FITC-Con A, SBA and WGA binding after preincubation with methyl-α- mannopyranoside, N-acetyl- galactosamine and N-acetyl- glucosamine respectively, to conidia from 18-d-old cultures

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Spore Middle Tip

400 200 0

IMI CH2 134523

CH1 B1300 A2 960 G8

A2 960 B1300 + N-acetyl-D glucosamine

Fig. 7

Germling fluorescent lectin avidity

Figs 5–7. Binding of FITC-conjugated Con A, SBA and WGA to germling regions. A l 95 % confidence limits for differences between regions within an isolate and B l 95 % confidence limits between isolates for a particular location.

Digital microscopy demonstrated that germlings had a marked avidity for Con A (Fig. 5) even though as already noted quantitative statistical comparison of test lectin avidity is precluded. An exposure period of only 200 ms sufficed to ensure full image bit depth of Con A. In contrast image exposure periods of 500 and 1250 ms were required to ensure full image bit depth of WGA and SBA treatments respectively, suggesting that germlings have greater avidity for WGA compared to SBA as potentially demonstrated by Figs 6–7. Although compared with other inhibitory haptens N-acetyl glucosamine appears somewhat less effective at blocking analogous lectin binding, Figs. 5–7 also demonstrate that lectin binding is specific, as preincubation of test lectins with an appropriate hapten significantly inhibited lectin binding by regions of representative strains (P 0n001). Fluorescent lectin binding to germlings showed significant variation in distribution, with the pattern of individual lectin binding appearing common to all trial isolates, except for the notable

case of G8 germling avidity for SBA. Lectins used were predominantly associated with a region of the germling. Fig. 8 shows that Con A bound in a relatively even manner over the entire hyphal surface of representative isolate CH2 but had greatest avidity for the spore walls of germlings, many of which fluoresced brightly and uniformly under illumination after FITC-Con A exposure. Quantitative fluorescence determinations confirm that Con A has significantly greater avidity for spore surfaces compared with those of tip and mid-hyphal regions (Fig. 5, P 0n001). Although this pattern of Con A binding is common to all strains investigated, individual strains differ in their Con A avidity with respect to location (P 0n001). In contrast SBA had least avidity for the residual spore walls of most test strains (Fig. 6) but greatest avidity for hyphal apices of germlings. Fig. 9 demonstrates SBA associated fluorescence of marked intensity around filament apices of

S. N. Smith and others

1537 particularly those reaches close to germling spore walls. In contrast to other test lectins conidial surfaces did not appear to bind fluorescent WGA in any uniform manner, as a range of lectin binding patterns may be observed including : even and bright fluorescence, bright but localized fluorescence or little fluorescence (Fig. 10). Mid-hyphal walls of all test strains bound significantly more WGA than hyphal tips or spore walls, even though as noted above, there is some variation amongst these latter entities (P 0n001), a pattern that only differed amongst strains in their individual avidity for WGA (Fig. 7, P 0n001).

5·0 lm

DISCUSSION

8 5·0 lm

9 5·0 lm

10 Figs 8–10. Microscopic appearance of germling binding of FITCconjugated Con A (isolate CH2) SBA (B1300) and WGA (CH2).

B1300, which then conspicuously declines with increasing distance from the hyphal tip. Such a distinct trend is shown by quantified hyphal tip and mid-hyphal fluorescence determinations (Fig. 6, P 0n001) to be common to the majority of trial isolates. Only one strain (G8) did not follow this pattern, which differed from the other isolates in its more uniform binding of fluorescent SBA (P 0n001). Fig. 10 demonstrates that WGA is not readily bound across apical regions of isolate CH2, however, fluorescent WGA was readily bound to the surface of mature hyphae throughout most of their length,

In this study flow cytometry and digital imaging have proven effective in determining the nature of fluorescent lectin binding to conidia and germlings of C. minitans. These respective procedures, when used in combination, have both the capacity to quantify lectin binding to populations of conidia and sufficient definition to characterise the surface distribution of fluorescent lectins. Although flow cytometry has been widely employed in studies of mammalian, microbial and to a lesser extent fungal spore populations (Allman, 1992 ; Shapiro, 1995 ; Davey & Kell, 1996) such investigations have concentrated on the light scattering properties of cells and their auto fluorescence. Reports of microbial fluorescent determinations are fewer (Davey & Kell, 1996) and focus primarily on bacterial and yeast populations. Results from this study, however, demonstrate that flow cytometry can also define the fluorescent lectin binding properties of conidia. Although differences in hyphal avidity for fluorescent lectins have been considered in detail (Freytag & Mendgen, 1991 ; O’Connell et al., 1996), such investigations have concentrated on distinct and separate morphological entities or infection structures rather than spatial distribution of lectins along short hyphal segments. This study, while quantifying lectin binding to germling regions, also demonstrates that digital imaging has the potential to characterize the spatial distribution of fluorescent lectins over continuous lengths of fungal filaments. There was no consistent relationship between colony type and lectin binding by conidia or germlings. Young conidia show the greatest avidity for the lectins utilized, so concomitantly must have the greatest numbers or optimum exposure of surface lectin receptors. Conidial populations of test C. minitans strains readily bind Con A in a homogenous manner and although fluorescent lectin probes may interact with hydrophobic moieties (Sachdev, Zodrow & Carubelli, 1979), the pattern of lectin binding with increasing conidial maturity does not match the hydrophobicity characteristics of similar strains (Smith et al., 1998). Furthermore, the significant reduction in Con A adhesion to conidial wall components, due to the presence of methyl-α- mannopyranoside, affirms the specific nature of Con A receptor affinity throughout conidial populations. The penchant of Con A for mannose or its glycosides (Doyle, 1994 : Van Damme et al., 1998) and, to a lesser extent glucose and N-acetylglucosamine residues, indicates that although these latter moieties may be present, C. minitans conidial walls in common with those of Beauveria bassiana conidia (Hegedus et al., 1992), but in contrast to

Conidial and germling lectin avidity propagules of rust fungi (Freytag & Mendgen, 1991), are potentially rich in mannose moieties which may even predominate. Conidial populations also show homogenous avidity for SBA, demonstrating that N-acetyl galactosamine is present at the surface of conidial walls, and, like mannose residues, uniformly distributed through population members. Although quantitative comparisons of relative lectin avidity is precluded due to differing specificities in fluorescence of lectin conjugates, N-acetyl galactosamine may be present or exposed in relatively small amounts compared with mannose moieties. Freytag & Mendgen (1991), Gold & Mendgen (1991) and O’Connell et al. (1996) demonstrated that fungal propagules readily bind WGA, but C. minitans conidia appear to bind only small quantities of this lectin, potentially indicative of either relatively little N-acetyl glucosamine and chitin in conidial walls, or the masking of these entities by other wall components. Furthermore, in contrast to the homogenous binding of Con A and SBA, flow cytometry results show that WGA binding differs amongst members of a conidial population, with a distinct sub-population showing greater avidity for this lectin. Members of such a sub-population may either have greater amounts of N-acetyl glucosamine receptors, a more favourable receptor orientation for lectin recognition, less extracellular matrix material masking lectin binding sites, or a combination of such elements. Conidial avidity for lectins appears to decrease in similar proportions with culture age, irrespective of lectin and strain. Such an observation suggests that receptors on conidia for the lectins tested either decay, thereby compromising receptor efficacy, or become masked, or lose optimum orientation as conidial walls age and mature. As lectin receptor interactions have a pivotal role in fungal host interaction (Tunlid, Jansson & Nordbring-Hertz, 1992 ; Bircher & Hohl, 1997) and potential hosts of C. minitans produce lectins with N-acetyl galactosamine specificity (Kellens et al., 1992), the loss of adhesin and lectin avidity, including those used in this study, may markedly compromise conidial adhesion of C. minitans as mediated by specific interactions and associated forces of hydrogen bonding (Marshall, 1991). Non-specific hydrophobic and electrostatic forces associated with conidia of C. minitans are also age dependent (Smith et al., 1998). Hence young C. minitans conidia may prove more potent propagules for biocontrol use than older conidia due to their greater hydrophobicity and lectin or adhesin binding properties. In comparison to conidia, germlings of C. minitans demonstrate a diverse range of lectin avidity, which varies with morphological region investigated. The germling spore walls of all strains bind significantly greater quantities of Con A, compared with other regions, confirming than mannose moieties may still predominate on the surface of conidia after their germination. Mature regions of germling hyphae have significantly greater avidity for WGA compared with other regions, indicating that the surface of hyphae are rich in Nacetyl glucosamine or chitin. Except for one strain, N-acetyl galactosamine appears abundant in hyphal tips, as binding of SBA is distinctly concentrated in such regions. However, the significantly lower levels of SBA binding by more mature hyphal regions of nearly all test strains, and their

1538 corresponding marked avidity for WGA and Con A, suggests that many N-acetyl galactosamine residues either decay or are masked by chitin microfibrils and outer wall extracellular matrices (Gooday, 1994) during wall maturation. Although Freytag & Mendgen (1991) and O’Connell et al. (1996) demonstrated that particular infection structures of fungal plant pathogens bound different lectins, the spatial orientation of lectin receptors amongst C. minitans germlings is potentially more subtle. The test lectins are bound by C. minitans germlings with markedly different avidity over near contiguous cell surfaces, furthermore such avidities are dynamic in nature altering with wall maturation. Specific adherence characteristics of hyphal surfaces could vary with an equivalent degree of subtlety, potentially influencing the stability and permanency of hyphal attachment to substrata or host structures in the case of mycoparasite interactions. Manocha, Chen & Rao (1990) and Inbar & Chet (1992) have demonstrated the involvement of lectins in mycopathogen interactions, however, further valuable insight could be obtained into fungal-fungal interaction mediated by lectins and adhesins. Digital microscopy, depending on availability of fluorescent lectins, has the potential to quantify cell surface modification, which may occur during mycopathogen-host interaction. In association with patch clamping (Garrill & Davies, 1994), digital microscopy may also be used to determine in real time individual cellular and intracellular response to the presence of a mycopathogen or potential host. REFERENCES Allman, R. (1992). Characterization of fungal spores using flow cytometry. Mycological Research 96, 1016–1018. Bircher, U. & Hohl, H. R. (1997). Surface glycoproteins associated with appressorium formation and adhesion in Phytophthora palmivora. Mycological Research 101, 769–775. Bourett, T. M., Czymmek, K. J. & Howard, R. J. (1998). An improved method for affinity probe localization in whole cells of filamentous fungi. Fungal Genetics and Biology 24, 3–13. Budge, S. P., McQuilken, M. P., Fenlon, J. S. & Whipps, J. M. (1995). Use of Coniothyrium minitans and Gliocladium virens for biocontrol of Sclerotinia sclerotiorum in glasshouse lettuce. Biological Control 5, 513–522. Davey, H. M. & Kell, D. B. (1996). Flow cytometry and cell sorting of heterogeneous microbial populations : the importance of single-cell analyses. Microbiological Reviews 60, 641–696. Doyle, R. J. (1994). Introduction to lectins and their interactions with microorganisms. In Lectin-Microorganism Interactions (ed. R. J. Doyle & M. Slifkin), pp. 1–65. Marcell Dekker : New York. Evenhuis, A., Verdam, B., Gerlagh, M. & Goosen-van de Geijn, H. M. (1995). Studies on major disease crops of caraway (Carum carvi) in the Netherlands. Industrial Crops and Products 4, 53–61. Freytag, S. & Mandgen, K. (1991). Carbohydrates on the surface of uredinospore- and basidiospore-derived infection structures of the heteroecious and autoecious rust fungi. New Phytologist 119, 527–534. Garrill, A. & Davies, J. M. (1994). Patch clamping fungal membranes : a new perspective on ion transport. Mycological Research 98, 257–263. Gold, R. E. & Mendgen, K. (1991). Rust basidiospore germlings and disease initiation. In The Fungal Spore and Disease Initiation in Plants and Animals (ed. G. T. Cole & H. C. Hoch), pp. 67–99. Plenum Press : New York. Gooday, G. W. (1994). Cell walls. In The Growing Fungus (ed. N. A. R. Gow & G. M. Gadd), pp. 45–62. Chapman & Hall : London. Hegedus, D. D., Bidochka, M. J., Miranpuri, G. S. & Khachatourians, G. G. (1992). A comparison of the virulence, stability and cell-wall-surface characteristics of three spore types produced by the entomopathogenic fungus Beauveria bassiana. Applied Microbiology and Biotechnology 36, 785–789.

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