Assessment of alcohol percentage test for fungal surface hydrophobicity measurement

Authors


Bing Cheng Si, University of Saskatchewan, College of Agriculture and Bioresources, Department of Soil Science, 51 Campus Dr., Saskatoon, SK S7N 5A8, Canada. E-mail bing.si@usask.ca

Abstract

Aim:  To determine whether assessing the penetration of solutions with different concentrations of ethanol (alcohol percentage test: APT) on fungal surfaces is effective in characterization of hydrophobicity on fungal surfaces.

Methods and Results:  APT and contact angle (CA) measurements were conducted on nine hydrophobic and two hydrophilic fungal strains from the phyla of Ascomycota, Basidiomycota and Zygomycota. There was a strong positive correlation (R2 = 0·95) between the APT and CA measurements from eight of the nine hydrophobic stains (four pathogenic and mycotoxigenic Fusarium taxa, one melanosporaceous biotrophic taxon, Alternaria sp, Penicillium aurantiogriseum and Cladosporium cladosporioides). Hydrophilic control strains, Mortierella hyalina and Laccaria laccata, had CAs <90° and no measurable degree of hydrophobicity using the APT method.

Conclusions:  The APT method was effective in measuring the degree of hydrophobicity and can be conducted on different zones of fungal growth.

Significance and Impact of the Study:  Characterization of fungal surface hydrophobicity is important for understanding of its particular role and function in fungal morphogenesis and pathogenesis. APT is a simple method that can be utilized for fungal hydrophobicity measurements when CA cannot be measured because of obscured view from aerial mycelia growth.

Introduction

Microbial cell walls and hydrophobicity of cell surfaces have recently been recognized for their importance in ecology, medicine, food industry, chemistry and biology. Surface hydrophobicity is crucial for several key functions, such as appressorium formation in the cells and also providing structure, shape, adhesion and aggregation (Lee and Dean 1994; Dague et al. 2007). These key functions may increase the virulence and pathogenicity of human and plant pathogenic micro-organism. Research into fungal surface hydrophobicity has increased dramatically in recent years because of the discovery of hydrophobins. Hydrophobins are proteins that are ubiquitous to filamentous fungi and are often implicated as one of the metabolites that contribute to fungal surface hydrophobicity (De Vries et al. 1993; Wessels 1994). These proteins are usually found and secreted on the outer surfaces of conidia, spores, aerial hyphae, infection structures and fruiting bodies (Wessels 1996; Kershaw and Talbot 1998; Wösten 2001; Linder et al. 2005). They also have important functions such as allowing the escape from aqueous environments, which then allows fungi to produce aerial mycelia and spores (Schuren and Wessels 1990; Linder et al. 2005).

Hydrophobicity mediates fungal attachment processes on other hydrophobic surfaces, which is an important step of fungal pathogenesis initiation (Howard and Valent 1996; Linder et al. 2005). Inhibition of this surface hydrophobicity might hinder fungal pathogenesis initiation (Tucker and Talbot 2001) and decrease aerial spore production, attachment, spread, pathogenicity and symbiotic association with a host (Talbot et al. 1993; Tucker and Talbot 2001). The degree of fungal hydrophobicity also has important implications for human health. The relationship between surface hydrophobicity and human pathogenic fungi is well documented in Candida albicans and Aspergillus fumigatus (Karkowska-Kuleta et al. 2009). Tighter adherence to epithelial cells, endothelial cells and extracellular proteins in Candida albicans as well as adhesion to albumin and collagen in A. fumigatus are the result of hydrophobic surface properties (Karkowska-Kuleta et al. 2009). Because of hydrophobic phenomena on fungal surfaces, characterization of this property is important for understanding its functions and role in the environment as well as the host.

Despite the noted importance of fungal surface hydrophobicity, characterization methods are limited to the manner in which the fungus grows. The majority of methods are based on indirect observations, such as growth behaviour on plates and broth cultures, excretions of hydrophobic compounds and aerial growth of mycelia and spore formation (Smits et al. 2003). Techniques used to assess microbial hydrophobicity are also subject to criticism, as most of these approaches are based on adhesion properties (Chau et al. 2009). Microbial adhesion techniques involve both electrostatic effects and hydrophobic binding interactions, which led to the conclusion that other factors may influence the results (Geertsema-Doornbusch et al. 1993; Doyle 2000). Also, adhesive methods involve manipulation of the specimen (washing, staining, extraction, adhesion and drying), which may drastically affect the hydrophobicity assessment through degradation of fungal cultures. To ensure accurate hydrophobicity assessment, the techniques employed must be direct and offer minimal disturbance to the culture.

Two simple techniques that are currently employed for characterization of hydrophobicity are the water drop penetration time (WDPT; Letey, 1969) and contact angle (CA) methods. WDPT measures the time taken by a water droplet to either penetrate or spread on a surface. Typically, WDPT is assessed on porous surfaces, but it is also applicable on planar surfaces by means of absorption (Unestam 1991). WDPT is a measure of the persistence of hydrophobicity on a particular surface. CAs refer to the angle of the liquid/vapour interface where a particular liquid meets the surface. It is also commonly known as a measurement of the degree of hydrophobicity. The acquisition of CAs is affected by the surface smoothness and uniformity, properties of the measuring liquids and the methodological approach. Unfortunately, because of the ability of fungi to produce aerial mycelia, CAs may be difficult to measure as the angle and baseline where it is measured is often obscured. In these certain instances, an additional method is required.

CA measurements and WDPT have been employed extensively in analyzing surface hydrophobicity and soil water repellency (Letey et al. 2000). However, Unestam (1991) proposed a technique similar to WDPT on fungal surfaces, by observing the absorption of 0·01-μl water droplets on ectomycorrhizal short roots, mycelia, rhizomorphs and mats. This allowed for a direct measure of persistence of hydrophobicity on fungal surfaces.

A liquid surface tension that has a surface CA of 90° was proposed as an index of water repellency by Watson and Letey (1970). This procedure is predicated on the assumption that a liquid can only completely wet a surface if the CA is <90°. For surfaces with a CA <90°, the surface tension of the droplet is assumed to be less than that of the surface. When the CA is >90°, surface tension of the droplet (liquid) is greater than that of the surface and thus will prevent wetting of the surface. Theoretically, a series of solutions providing various surface tensions can be prepared and placed onto a hydrophobic surface. The solutions with higher surface tension will tend to reside on the surface and contribute to a higher degree of hydrophobicity, while solutions with lower surface tension will either penetrate or spread on the surfaces because of a lower degree of hydrophobicity. As discussed earlier, surface tension with a CA equal to 90° is the surface tension of a solution where there is a transition from wetting, to repelling on the surface. Fluids with low surface tension can be mixed with miscible fluids that have high surface tension to create a series of solutions with varying surface tensions. Alcohol percentage test (APT) (Dekker and Ritsema 1994), more commonly referred to as the molarity of ethanol method (MED) (Watson and Letey 1970) was developed based on the fact that ethanol has a smaller surface tension (0·0219 N m−1) than water (0·0719 N m−1). APT uses aqueous ethanol solutions with different concentrations to determine the lowest concentration of ethanol solution that absorbs or wets the surface (Watson and Letey 1970). The higher the ethanol concentration that wets the surface, the more severe the degree of hydrophobicity is. Five and 10 s are commonly utilized as the reference time for absorption or wetting (Letey et al. 2000). Application of this technique on fungal surfaces has yet to be assessed and may offer another direct measure of the degree of hydrophobicity. However, this approach on fungi may present some challenges because of the delicate nature of fungi as it may cause degradation of its hydrophobicity. As such, a reference point of approx. 5-s or less is more reasonable for assessing hydrophobicity on fungal surfaces because of the effect of hydrophobicity degradation (Crockford et al. 1991). The objective of this study was to determine whether APT is an effective approach to characterize the hydrophobicity on fungal surfaces.

Materials and methods

Fungal strains

Eleven fungal strains from phyla of Ascomycota, Basidiomycota and Zygomycota were selected for assessing the application of the APT method. Four Fusarium strains (Fusarium avenaceum, Fusarium oxysporum, 3- and 15-acetyldeoxynivalenol-producing Fusarium graminearum chemotypes), one biotrophic mycoparasite –Sphaerodes mycoparasitica SMCD 2020, Alternaria sp. (Kunze) Wiltshire SMCD 2122, Penicillium aurantiogriseum Dierckx SMCD 2151, Cladosporium cladosporioides (Fresen.) G.A. de Vries SMCD 2128, Cladosporium minourae Iwatsu SMCD 2130, Mortierella hyalina (Harz), W. Gams SMCD 2145 and Laccaria laccata Scop & Cooke UAMH 10033/SMCD 2265, were obtained from Saskatchewan Microbial Collection and Database (SMCD) and University of Alberta Microfungus Collection and Herbarium (UAMH). The fungal isolates were maintained on potato dextrose agar (Difco) supplemented with antibiotics (100 mg l−1 streptomycin sulfate and 12 mg l−1 neomycin sulfate) (Sigma–Aldrich) prior to the experiments.

Contact angles

Fungal cultures were inoculated onto slide media (Chau et al. 2009) and were incubated in the dark at 23°C. Growth was assessed daily until complete coverage of the glass slide was observed (Table 1). Approximately five to ten 10 μl droplets of water were deposited onto fungal surfaces. Pictures were taken immediately following deposition of the droplets. CAs of the droplets were measured by obtaining the images using a modified microscopy apparatus and fitting a drop profile using Low Bond Axisymmetric Drop Shape Analysis Model of Drop Shape Analysis (LB_ADSA) (Stalder et al. 2006; Chau et al. 2009). Fungal plates were prepared in triplicates, while the experiment was repeated twice. CAs obtained on slide cultures were used to validate the APT method by linear regression.

Table 1.   The age of cultures for complete coverage of slide media and contact angles obtained from surface measurements
Fungus culturesTime (days)PDA* (θw)
  1. *Potato dextrose agar media.

Fusarium avenaceum7108° ± 3
Fusarium oxysporum7116° ± 1
3-Acetyldeoxynivalenol Fusarium graminearum7124° ± 1
15-Acetyldeoxynivalenol F. graminearum7125° ± 2
Sphaerodes mycoparasitica7125° ± 1
Cladosporium cladosporioides10142° ± 1
Cladosporium minourae10142° ± 5
Penicillium aurantiogriseum10128° ± 1
Alternaria sp.5122° ± 1
Laccaria laccata300° ± 0
Mortierella hyalina759° ± 1

Alcohol percentage test

A series of aqueous ethanol solutions were prepared in 5% increments starting from 0 to 100% ethanol (Dekker and Ritsema 1994). Conversions of alcohol percentages to molarity, or surface tension, can be performed by using the relationship illustrated in Watson and Letey (1970) or refer to previous published data in Butler and Wightman (1932) and Roy and McGill (2002). The APT/MED protocol described by Watson and Letey (1970) and Crockford et al. (1991) was used. Four-microlitre droplets of ethanol solutions were applied on the surface of fungal colonies, and the time interval used for infiltration of the solution droplets was <5-s. This short penetration time was vital to ensure that hydrophobicity decay did not affect our results (Crockford et al. 1991). Inner and outer zones of fungal colony growth were defined by observations of two distinct zones, with differences in colour, structure and aerial mycelia. Replicates of three droplets on each zone were assessed on three replicates of fungal cultures.

Results

Laccaria laccata and M. hyalina were the control strains and showed no hydrophobicity with CAs <90°, resulting in an APT reading of zero (Fig. 1a). Strains with the highest CA were Cladosporium (>140°), while F. avenaceum strains had the lowest CA (108°) among all hydrophobic fungal strains (Fig. 1a). This APT reading (degree of hydrophobicity) was because of surface tension of the fungus (>0·0719 joules m−2) being greater than that of any of the aqueous ethanol solutions.

Figure 1.

 (a) Plot of fungal strains vs the alcohol percentage (ethanol) absorbed into fungal surfaces on potato dextrose agar (PDA)-grown cultures and contact angles (CA) of water droplets on the fungal inoculated PDA slides (Chau et al. 2009): (•) CAs; (▪) inner zone alcohol percentage test (APT) (%) and (inline image) outer zone APT (%). Note: Strains designated as Laccaria laccata (LL), Mortierella hyalina(MH), Fusarium avenaceum (FH), Fusarium oxysporum (FA), Alternaria sp. (A), Cladosporium minourae (CM), Sphaerodes mycoparasitica (SA), 3-acetyldeoxynivalenol Fusarium graminearum (3A-FG), 15-acetyldeoxynivalenol F. graminearum (15A-FG), Penicillium aurantiogriseum (PA) and Cladosporium cladosporioides (CC). (b) The relationship between the ethanol percentage method and water CA measurements on agar inoculated hydrophobic fungal cultures (fitted by linear regression R2 = 0·95): (•) Fungal strains and (○) C. minourae. *Removed from linear correlation because of interaction effects from ethanol solutions.

Through characterization of hydrophobicity using the APT method, we found that Alternaria sp., C. cladosporioides, C. minourae and 15-acetyldeoxynivalenol-producing F. graminearum chemotype exhibited two zones of hydrophobicity. It was observed that mycelial growth further from the point of inoculation showed growth into the media; while in the centre aerial mycelia were being produced. However, it was noted that 15-acetyldeoxynivalenol-producing F. graminearum chemotype showed higher degrees of hydrophobicity on white/aerial mycelia (APT = 73 ± 3%) than the red pigment growth in the centre (APT = 55 ± 0%) (Fig. 1a). The red pigment growth eventually replaced the growth of the white mycelia.

Linear relationships between CA and APT between hydrophobic strains were low (R2 = 0·50) and mainly because of one of the fungal strains, that is, C. minourae. Indeed, removal of this strain resulted in a better correlation (R2 = 0·95) (Fig. 1b). APT values for C. minourae of 45% may be attributed to some interaction between the ethanol and fungal surfaces or the effect of ethanol on fungal cultures.

Discussion

Results presented in this study revealed differential expressions of fungal hydrophobicity, which may be based on the age of mycelia growth. Understanding the expression pattern of fungal hydrophobicity may further provide insight into its implications, such as prepathogenesis in plant pathogenic fungi and attachment to host in human pathogenic fungi (Tucker and Talbot 2001; Karkowska-Kuleta et al. 2009). Wessels et al. (1991) found that accumulation of hydrophobins on older mycelia growth when compared to younger growth may cause a decrease in hydrophobicity assessment at the location further away from the point of inoculation. Differences in the zones of growth may also be because of depletion of nutrients at the point of inoculation, resulting in a shift from primary to secondary metabolism (Smits et al. 2003). As we know, submerged and aerial hyphae have been shown to have differences in functions and surface hydrophobicity (Wösten and Willey 2000). Fungi tend to acquire their nutrients by hydrophilic means (Unestam 1991). However, when nutrients are depleted, formation of hydrophobic aerial structures and spore productions will occur. Distinguishing the differences in aerial and submerged hyphae hydrophobicity may aid in understanding spore dispersal patterns of certain types of fungi (Wösten and Willey 2000). The APT method allows for the assessment of hydrophobicity on these two morphologically or chemically different regions because this method is a direct measurement and requires very little solution (<10 μl).

The 15-acetyldeoxynivalenol-producing F. graminearum chemotype showed a higher degree of hydrophobicity on white/aerial mycelia than the red growth in the centre. This could be because of the generation of more soluble toxin or pigment compounds when compared to white aerial mycelia growth. In several previous studies, few Fusarium mycotoxins were found to be more hydrophobic then others (Yoshizawa and Morooka 1973; Elosta et al. 2007). Red pigmentation is related to melanin or aurofusarin production and might offer protection against desiccation stress (Butler and Day 1998). However, Prota (1992) found that melanin contains large amounts of water to preserve the structure of the pigment. The ability of melanin to store water and ions may suggest lower degrees of hydrophobicity when compared to the aerial mycelia (White 1958). More research is needed to better understand the importance of morphological and hydrophobic regions as well as their relationship with Fusarium function and pathogenicity.

Furthermore, additional research is needed to examine how hydrophobicity will change because of alteration of growth conditions, such as nutrient status, pesticide and chemical treatment, and the presence of host plant and biocontrol organisms such as bacteria. In the case of altering growth conditions, there is the potential for alteration of fungal surface hydrophobicity in response to the changes (Smits et al. 2003; Feeney et al. 2006;Chau et al. 2009). It is also possible that any changes that may occur will result in a decrease in pathogenicity of plant pathogens (Talbot et al. 1993; Kazmierczak et al. 2005).

Hydrophilic classification of fungi is not suitable from APT measurements, because of the fact that surface tensions of fungal surfaces are always lower than the tension of aqueous ethanol solutions. Therefore, the result is an instantaneous infiltration or spreading of the solution. With the current limitations of the CA approach, related to its subjective nature and the possibility of obscured views, the APT method may offer an alternative approach for characterization of fungal hydrophobicity. This study has shown that the APT method is useful for analyzing the degree of hydrophobicity of hydrophobic fungal strains through comparison of CA measurements on the fungal surfaces. APT measurements were also useful for characterization of different zones of hydrophobicity on the same culture. This will aid in providing a better understanding of the expression patterns of hydrophobicity in zones of colour changes and aged growth because of different morphological, chemical, or metabolic reasons. Given the advantages of the APT method such as the reproducibility and simplicity, it should be considered as one of the methods for quantifying the degree of hydrophobicity on fungal surfaces.

Acknowledgements

This research was financially supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and Agri-Food Innovation Found Chair (AFIF) in Mycology, Microbial Biotechnology & Bioproducts to Dr V. Vujanovic, Dean Scholarship to H.W.C and Departmental Devolved Scholarship to Y.K.G. We thank Lindsay Tallon from The School of Environment and Sustainability, University of Saskatchewan for helpful discussion and review of this manuscript.

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