Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Effects of biosurfactants, mannosylerythritol lipids, on the hydrophobicity of solid surfaces and infection behaviours of plant pathogenic fungi S. Yoshida1,†, M. Koitabashi1,†, J. Nakamura2, T. Fukuoka3, H. Sakai2, M. Abe2, D. Kitamoto3 and H. Kitamoto1 1 Biofunction Division, National Institute for Agro-Environmental Sciences, Tsukuba, Japan 2 Faculty of Science and Technology, Tokyo University of Science, Noda, Japan 3 Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
Keywords anthracnose, Blumeria graminis f. sp. tritici, Colletotrichum dematium, Glomerella cingulata, Magnaporthe grisea, mannosylerythritol lipids, powdery mildew, rice blast. Correspondence Hiroko Kitamoto, Biofunction Division, National Institute for Agro-Environmental Sciences, 3-1-3, Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan. E-mail: [email protected] †
The first two authors contributed equally to this work and should be considered co-first authors. 2015/0177: received 27 January 2015, revised 6 April 2015 and accepted 18 April 2015 doi:10.1111/jam.12832
Abstract Aims: To investigate the effects of mannosylerythritol lipids (MELs) on the hydrophobicity of solid surfaces, their suppressive activity against the early infection behaviours of several phytopathogenic fungal conidia, and their suppressive activity against disease occurrences on fungal host plant leaves. Methods and Results: The changes in the hydrophobicity of plastic film surfaces resulting from treatments with MEL solutions (MEL-A, MEL-B, MELC and isoMEL-B) and synthetic surfactant solutions were evaluated based on the changes in contact angles of water droplets placed on the surfaces. The droplet angles on surfaces treated with MELs were verified to decrease within 100 s after placement, with contact angles similar to those observed on Tween 20-treated surfaces, indicating decreases in surface hydrophobicity after MEL treatments. Next, conidial germination, germ tube elongation and the formation of appressorium of Blumeria graminis f. sp. tritici, Colletotrichum dematium, Glomerella cingulata and Magnaporthe grisea were evaluated on plastic surfaces that were pretreated with surfactant solutions. On the surfaces of MEL-treated plastic film, inhibition of conidial germination, germ tube elongation, and suppression of appressoria formation tended to be observed, although the level of effect was dependent on the combination of fungal species and type of MEL. Inoculation tests revealed that the powdery mildew symptom caused by B. graminis f. sp. tritici was significantly suppressed on wheat leaf segments treated with MELs. Conclusions: MELs exhibited superior abilities in reducing the hydrophobicity of solid surfaces, and have the potential to suppress powdery mildew in wheat plants, presumably due to the inhibition of conidial germination. Significance and Impact of the Study: This study provides significant evidence of the potential for MELs to be used as novel agricultural chemical pesticides.
Introduction Microbial surfactants, generally referred to as biosurfactants, possess unique surface-active properties and highly diverse structures (Cooper and Zajic 1980), and have attracted attention as alternatives to synthetic surfactants due to their greater biodegradability and safety (Kitamoto
et al. 2002; Kitamoto et al. 2009). Among them, mannosylerythritol lipids (MELs), which consist of 4-O-b-Dmannopyranosyl-erythritol as a hydrophilic moiety and acetyl groups and fatty acids as hydrophobic moieties, are one of the better-characterized glycolipid biosurfactants (Banat et al. 2010) produced by fungal genera, such as Pseudozyma, Ustilago and Schizonella yeasts (Arutchelvi
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et al. 2008). MELs produced in the culture filtrates of Pseudozyma species mainly consist of MEL-A, MEL-B and MEL-C, which are distinguished based on the numbers and positions of acetyl groups in their chemical structures, and their compositions are species- and/or strain-specific in the genus (Konishi et al. 2007; Fukuoka et al. 2008). Furthermore, an unusual MEL-B (a diastereomer type of MEL-B: isoMEL-B) with a carbohydrate structure different from that of conventional MEL-B has been isolated from Pseudozyma tsukubaensis (Fukuoka et al. 2008). Because this MEL family has specific and superior interfacial properties (Kitamoto et al. 2002), it has been utilized in various emulsifiers, dispersants, and washing detergents as well as anti-agglomeration agents for ice-slurry (Kitamoto et al. 2001) and as ingredients for skin care products (Yamamoto et al. 2012; Morita et al. 2013). Although MELs have been utilized in various industrial and medical applications due to their specific properties, no practical application in agriculture has been developed. One promising applicable usage is as pesticides against phytopathogenic fungi on plant surfaces. Several plant pathogenic fungi infecting aboveground plant parts cause diseases through early infection behaviours, which consist of conidial germination and development and elongation of germ tubes, followed by appressorium formation (Preece and Dickinson 1971). Although there are various factors that influence each infection behaviour, hydrophobicity of plant surfaces has been identified as an inducing factor (Lee and Dean 1994; Hegde and Kolattukudy 1997; Chaky et al. 2001; Tsuba et al. 2002; Shaw et al. 2006; Zabka et al. 2008). Changes in plant surface hydrophobicity are therefore hypothesized to inhibit fungal behaviour on the surface, leading to suppression of disease occurrences. Recently, we reported that MEL solutions had good wetting ability on several plant leaves (Fukuoka et al. 2015), which indicates the potential of the solutions to reduce the hydrophobicity of the surface. Inhibition of early infection behaviours by a biosurfactant has been reported for flocculosin, a glycosyl erythritol type biosurfactant produced by Pseudozyma flocculosa, which causes bursting of conidia of Erysiphe graminis f. sp. tritici (Hajlaouia and Belangera 1993; Hammami et al. 2011). However, to date, no such inhibitory roles of MELs against initial fungal behaviours have been elucidated, although several studies demonstrating the direct antimicrobial activities of compounds against several micro-organisms, such as Gram-positive bacteria (Kitamoto et al. 1993) and phytopathogenic fungi (Ishii et al. 2011), have been reported. The first objective of this study was to verify the ability of MELs to decrease the hydrophobicity of solid surfaces by comparing them to several commercial synthetic surfactants currently in use. Among the members of the MEL 216
family, we focused on MEL-A, MEL-B, MEL-C and isoMEL-B, which are all di-acylated MELs, because their physiochemical properties have been well characterized, and information about processes relating to their massproduction are available (Arutchelvi et al. 2008; Fukuoka et al. 2008). IsoMEL-B is now commercially available from TOYOBO Co. Ltd. (Osaka, Japan). After demonstrating that MELs changed the hydrophobicity of the surfaces, we verified the effects of MEL treatments on the early infection behaviours of representative phytopathogenic fungal conidia, as well as their suppressive activities against disease occurrences on host leaves caused by the fungal conidia. As model plants, we used the leaves of two Gramineae plants (i.e. wheat, rice), the surfaces of which are highly hydrophobic (Watanabe and Yamaguchi 1991), and the leaves of two non-Gramineae plants (i.e. strawberry and mulberry), which are presumably more hydrophilic than those of the former plant family (Watanabe and Yamaguchi 1991; Fukuoka et al. 2015). Materials and methods Materials The surfactants used in this study are members of the MEL family, MEL-A, MEL-B, MEL-C and isoMEL-B, which are produced by various species of Pseudozyma yeast strains (Morita et al. 2007; Fukuoka et al. 2008). Each MEL was isolated and purified from culture filtrates of the Pseudozyma strains, as described previously (Kitamoto et al. 1990a; Morita et al. 2007; Fukuoka et al. 2008). As synthesized surfactants, polyoxyethylene (20) sorbitan monolaurate (Tween 20) (Wako chemical, Tokyo, Japan) and polyoxyethylene (10) lauryl ether (Brij 35) (Wako chemical) were also used. The surfactants were individually adjusted to 01% (w/w) concentrations in sterilized distilled water (SDW), and were used for the following experiments after standing for at least 24 h. A MEL mixture (01%) extracted from the culture broth of Pseudozyma antarctica T-34 that contained MEL-A, MELB and MEL-C (mixing ratio, 58 : 25 : 10) (Kitamoto et al. 1990b) was alternatively used for each compound in a disease inhibition assay against Magnaporthe grisea, because preliminary experiments using each MEL compound did not inhibited the disease (data not shown). All of the reagents and solvents were commercially available and used as received. Measurement of contact angles of water droplets on plastic surfaces pretreated with surfactants The droplet contact angles (h) of water droplets on abiotic surfaces were evaluated based on Young’s equation
Journal of Applied Microbiology 119, 215--224 © 2015 The Society for Applied Microbiology
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(Adamson and Gast 1997); for droplets placed on a surface, smaller values of h indicate stronger attraction to the solid surface. As the abiotic surface, small segments (2–3 cm2) of plastic film (Gelbond: Takara, Otsu, Japan) were used for the measurements. The film was immersed in a surfactant solution or in SDW as a control. After air-drying, an SDW droplet (10 ll) was placed onto the hydrophobic side of the film segment, and the h value of the droplet at 100 s after placement was measured using Drop Master DM500 (Kyowa Interface Science Co., LTD, Niiza, Japan). Measurements were independently repeated in triplicate, and the obtained data were averaged and then subjected to ANOVA followed by Scheffe’s test using the KALEIDAGRAPH 4.1 J software (HULINKS Inc., Tokyo, Japan). Fungal isolates Wheat powdery mildew fungus, Blumeria graminis f. sp. tritici strain T-10 (hereafter Bg), mulberry anthracnose fungus, Colletotrichum dematium strain S9733 (hereafter Cd), strawberry anthracnose fungus, Glomerella cingulata strain S0709 (hereafter Gc) and rice blast fungus, M. grisea strain Kyu89–246 (hereafter Mg), all of which were preserved in our laboratory, were used in this study. Preparation of fungal inocula Conidia of the obligate parasitic fungus, Bg, were prepared according to the shaking method (Moseman et al. 1965). Briefly, potted wheat seedlings (cultivar (cv.) Ayahikari) grown in a glass house and artificially infested with Bg were periodically maintained by shaking off the conidia, thereby presenting the symptom to new healthy seedlings; the diseased seedlings harbouring fresh symptoms and conidia on the plant surface were used as the inoculum. The conidial suspension of Cd was prepared according to the method previously reported by Yoshida et al. (2000). Briefly, conidia of Cd harvested from 10day-old mulberry leaf decoction sucrose agar (MSA: dried mulberry leaf, 50 g; sucrose, 30 g; agar, 27 g; distilled water, 15 l) were washed twice in SDW by centrifugation (6000 g for 5 min), and then adjusted to a concentration of 5 9 105 conidia ml 1 using a hemocytometer. To prepare the conidial suspension of Gc, the fungal strain was shake-cultured (100 rev min 1) in a 200 ml Erlenmeyer flask containing 30 ml potato dextrose broth (Difco, Detroit, MI) at 25°C for 5 days. Conidia in the culture were harvested by pouring the incubation mixture through cotton mesh, followed by centrifugation (6000 g for 5 min). The harvested conidia were washed twice by centrifugation and adjusted to a concentration of 5 9 105 conidia ml 1, as described above. Conidia of Mg
MELs on hydrophobicity and fungal infection
were produced on the surface of colonies of oatmeal agar (Difco) plates and incubated at 25°C under 12 h black light (BLB) light:12 h dark conditions, harvested in SDW with an artist’s brush, and then adjusted to a concentration of 5 9 105 conidia ml 1, as described above. Observation of conidial germination and appressorial formation on hydrophobic plastic surfaces pretreated with surfactants To observe the effects of surface hydrophobicity attributed to surfactants against conidial germination, lengths of germ tubes and appressorial formation, 10 ll of each surfactant solution were placed on the hydrophobic surface of 10 Gelbond film segments (approx. 2 cm2) and then air-dried. Untreated segments were also prepared as a control. Subsequently, aliquots (5 ll) of the conidial suspensions of each fungus, with the exception of Bg, prepared as described above, were placed onto the film segments treated with surfactants. To analyze Bg, conidia appearing on diseased wheat seedlings were sprinkled onto the surface of each surfactant-treated Gelbond segment by the shaking method (Moseman et al. 1965), and approx. 90 conidia mm 2 were attached to the surface. The segments were then incubated in a moist plastic chamber at 25°C in the dark for 1 day, and 2–3 ll lactophenol cotton blue reagent (phenol, 10 g; lactic acid, 10 g; glycerol, 20 g; distilled water, 10 g; cotton blue, 0025 g) were added to each droplet on the segments to stain and fix fungal tissues, after which the droplets were mounted with cover slips. Fungal morphologies (ratios of conidial germination, lengths of germ tubes, ratios of appressorial formation) on each segment were observed using a light microscope (Eclipse 80i; Nikon, Tokyo, Japan). The ratio of conidial germination was determined by observing 50 randomly chosen conidia of each fungal strain. For evaluation of germ tubes lengths, the degrees of the lengths of germinated conidia were determined based on the length relative to that of the main axis of the conidia with the following index values: 1, ≤1-fold; 2, >1-fold to 2-fold; 3, >2-fold to 4-fold; 4, >4-fold. The ratio of appressorial formation was evaluated by observing the formation of typical pigmented appressoria at the tips of germ tubes of the germinated conidia of each fungal strain. These observations were carried out in triplicate independently, and the averaged data were subjected to ANOVA followed by Dunnett’s test using the KALEIDAGRAPH 4.1 J software. Disease suppression assay For each surfactant solution, after treatment on the surface of leaves, suppressive activity against symptom
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Results Contact angles of water droplets on plastic surfaces pretreated with surfactants The initial averaged h value of SDW droplets on the SDW-treated plastic film was 813° (data not shown), and the value at 100 s after placement was 763° (Fig. 1). Under such hydrophobic surface conditions, h values of SDW droplets on surfactant solution-treated surfaces were verified to decrease at 100 s after dropping (Fig. 1). The droplet angles at 100 s on surfaces treated with MEL-A and isoMEL-B were below 55 and 70°, respectively, and their angles were not significantly different
Contact angle of droplet (θ )
80°
c
60°
40°
20°
er
35
at
W
ij
Br
n ee 20
-B
EL
oM
-C
-A
EL
Tw
M
Is
N.D.
-B EL
N.D. M
0°
b
a
a
a
EL
218
index values 0–6, representing the ratio of lesion area (0, no lesion; 1, trace appearance; 2, ≤5%; 3, >5 to 10%; 4, >10 to 25%; 5, >25 to 50%; 6, >50%), on three randomly chosen mature leaves per seedling. Disease suppression in each inoculation test was evaluated based on the averaged values among the triplicated data, and the averages were subjected to ANOVA followed by Dunnett’s test as described above.
M
development of powdery mildew on wheat leaves from Bg, anthracnose of mulberry and strawberry from Cd and Gc, respectively, and rice blast from Mg, was evaluated. For the assay against powdery mildew of wheat, 10 cm long primary leaves of potted wheat seedlings (cv. Ayahikari) grown in the glass house as described above were cut out. The leaf segments were then immersed in each surfactant solution in a test tube. After the leaves were air-dried, conidia of Bg on diseased wheat leaves were sprinkled onto the surfaces of the treated leaves by the shaking method, as described above. The inoculated leaves were incubated in moist chambers for 7 days at 20°C under 12 h light: 12 h dark conditions. The degree of necrotic symptom appearance was evaluated based on lesion index values 0–4, representing the ratio of the lesion area on each leaf (0, no lesion; 1, ≤10%; 2, >10 to 25%; 3, >25 to 75%; 4, >75%). Five leaf replicates were used for each surfactant treatment. In the suppressive assay against mulberry anthracnose from Cd, small segments (approx. 15 cm2) of mature healthy mulberry leaves detached from a mulberry tree (unknown cultivar) planted on the grounds of National Institute for AgroEnvironmental Sciences, Tsukuba, were prepared. Aliquots (10 ll) of each surfactant solution were placed on the adaxial surfaces of individual segments. After the solutions were air-dried, 5 ll of conidial suspension (105 conidia ml 1), prepared as described above, were placed onto the surfaces of the treated segments. Suppressive activity resulting from treatment was evaluated based on the appearance and diameter (mm) of the round and brown lesions on leaf segments after 7 days of incubation in a moist chamber at 25°C in the dark. Seven replicate leaf segments were used for each surfactant treatment. The experiment against strawberry anthracnose caused by Gc was performed using segments (approx. 15 cm2) of strawberry leaves (cv. Nyohou) dissected from potted nurseries grown in a glass house. Treatments of surfactant solutions, inoculation of the conidial suspension (105 conidia ml 1), and evaluation of suppressive activity were performed in the same manner as the experimental treatment of mulberry leaves against Cd. For suppressive activity against rice blast disease caused by Mg, 9–10 rice seedlings (cv. Koshihikari) at the fourth–fifth leaf stage in a plastic nursery box (15 9 6 9 10 cm) grown in a glass house were used. A MEL mixture (20 ml; a substitute for MEL-A, MEL-B and MEL-C), Tween 20 and Brij 35, were sprayed onto the seedlings in the nursery box, and then air-died. Then, 20 ml of conidial suspension of Mg (5 9 105 conidia ml 1) was similarly sprayed onto the seedlings. The inoculated seedlings were incubated in a moist chamber for 2 days at 25°C, and then kept in a glass house for 5 days at 25°C. Subsequently, the degree of symptom appearance was evaluated based on lesion
Surface treatment Figure 1 Contact angles of sterilized distilled water droplets on plastic (Gelbond) surfaces pretreated with surfactant solutions at 100 s after placement. The data represent averages from three independent experiments, and bars indicate the standard error of the mean. ND indicates that measurements of angles were not determined. The same letter above multiple columns indicates that the values were not significantly different according to Scheffe’s test (P < 005).
Journal of Applied Microbiology 119, 215--224 © 2015 The Society for Applied Microbiology
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MELs on hydrophobicity and fungal infection
from those on the Tween 20-treated surfaces (h = 68), whereas droplets on the Brij 35 (h = 93) and water-treated surfaces exhibited significantly (P < 005) larger angles. Because droplets on the MEL-B and MEL-C-treated surfaces immediately spread over the limit of detection, their h values at 100 s were not determined. Effects of surfactant treatments on conidial germination and lengths of germ tubes on hydrophobic plastic surfaces Figure 2 shows the ratios of conidial germination of the examined fungal isolates on the surfactant-treated Gelbond film segments. Against Bg conidial germination, treatments of MEL-A and isoMEL-B significantly (P < 005) suppressed germination, with a germination ratio of
ORIGINAL ARTICLE
Effects of biosurfactants, mannosylerythritol lipids, on the hydrophobicity of solid surfaces and infection behaviours of plant pathogenic fungi S. Yoshida1,†, M. Koitabashi1,†, J. Nakamura2, T. Fukuoka3, H. Sakai2, M. Abe2, D. Kitamoto3 and H. Kitamoto1 1 Biofunction Division, National Institute for Agro-Environmental Sciences, Tsukuba, Japan 2 Faculty of Science and Technology, Tokyo University of Science, Noda, Japan 3 Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
Keywords anthracnose, Blumeria graminis f. sp. tritici, Colletotrichum dematium, Glomerella cingulata, Magnaporthe grisea, mannosylerythritol lipids, powdery mildew, rice blast. Correspondence Hiroko Kitamoto, Biofunction Division, National Institute for Agro-Environmental Sciences, 3-1-3, Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan. E-mail: [email protected] †
The first two authors contributed equally to this work and should be considered co-first authors. 2015/0177: received 27 January 2015, revised 6 April 2015 and accepted 18 April 2015 doi:10.1111/jam.12832
Abstract Aims: To investigate the effects of mannosylerythritol lipids (MELs) on the hydrophobicity of solid surfaces, their suppressive activity against the early infection behaviours of several phytopathogenic fungal conidia, and their suppressive activity against disease occurrences on fungal host plant leaves. Methods and Results: The changes in the hydrophobicity of plastic film surfaces resulting from treatments with MEL solutions (MEL-A, MEL-B, MELC and isoMEL-B) and synthetic surfactant solutions were evaluated based on the changes in contact angles of water droplets placed on the surfaces. The droplet angles on surfaces treated with MELs were verified to decrease within 100 s after placement, with contact angles similar to those observed on Tween 20-treated surfaces, indicating decreases in surface hydrophobicity after MEL treatments. Next, conidial germination, germ tube elongation and the formation of appressorium of Blumeria graminis f. sp. tritici, Colletotrichum dematium, Glomerella cingulata and Magnaporthe grisea were evaluated on plastic surfaces that were pretreated with surfactant solutions. On the surfaces of MEL-treated plastic film, inhibition of conidial germination, germ tube elongation, and suppression of appressoria formation tended to be observed, although the level of effect was dependent on the combination of fungal species and type of MEL. Inoculation tests revealed that the powdery mildew symptom caused by B. graminis f. sp. tritici was significantly suppressed on wheat leaf segments treated with MELs. Conclusions: MELs exhibited superior abilities in reducing the hydrophobicity of solid surfaces, and have the potential to suppress powdery mildew in wheat plants, presumably due to the inhibition of conidial germination. Significance and Impact of the Study: This study provides significant evidence of the potential for MELs to be used as novel agricultural chemical pesticides.
Introduction Microbial surfactants, generally referred to as biosurfactants, possess unique surface-active properties and highly diverse structures (Cooper and Zajic 1980), and have attracted attention as alternatives to synthetic surfactants due to their greater biodegradability and safety (Kitamoto
et al. 2002; Kitamoto et al. 2009). Among them, mannosylerythritol lipids (MELs), which consist of 4-O-b-Dmannopyranosyl-erythritol as a hydrophilic moiety and acetyl groups and fatty acids as hydrophobic moieties, are one of the better-characterized glycolipid biosurfactants (Banat et al. 2010) produced by fungal genera, such as Pseudozyma, Ustilago and Schizonella yeasts (Arutchelvi
Journal of Applied Microbiology 119, 215--224 © 2015 The Society for Applied Microbiology
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S. Yoshida et al.
et al. 2008). MELs produced in the culture filtrates of Pseudozyma species mainly consist of MEL-A, MEL-B and MEL-C, which are distinguished based on the numbers and positions of acetyl groups in their chemical structures, and their compositions are species- and/or strain-specific in the genus (Konishi et al. 2007; Fukuoka et al. 2008). Furthermore, an unusual MEL-B (a diastereomer type of MEL-B: isoMEL-B) with a carbohydrate structure different from that of conventional MEL-B has been isolated from Pseudozyma tsukubaensis (Fukuoka et al. 2008). Because this MEL family has specific and superior interfacial properties (Kitamoto et al. 2002), it has been utilized in various emulsifiers, dispersants, and washing detergents as well as anti-agglomeration agents for ice-slurry (Kitamoto et al. 2001) and as ingredients for skin care products (Yamamoto et al. 2012; Morita et al. 2013). Although MELs have been utilized in various industrial and medical applications due to their specific properties, no practical application in agriculture has been developed. One promising applicable usage is as pesticides against phytopathogenic fungi on plant surfaces. Several plant pathogenic fungi infecting aboveground plant parts cause diseases through early infection behaviours, which consist of conidial germination and development and elongation of germ tubes, followed by appressorium formation (Preece and Dickinson 1971). Although there are various factors that influence each infection behaviour, hydrophobicity of plant surfaces has been identified as an inducing factor (Lee and Dean 1994; Hegde and Kolattukudy 1997; Chaky et al. 2001; Tsuba et al. 2002; Shaw et al. 2006; Zabka et al. 2008). Changes in plant surface hydrophobicity are therefore hypothesized to inhibit fungal behaviour on the surface, leading to suppression of disease occurrences. Recently, we reported that MEL solutions had good wetting ability on several plant leaves (Fukuoka et al. 2015), which indicates the potential of the solutions to reduce the hydrophobicity of the surface. Inhibition of early infection behaviours by a biosurfactant has been reported for flocculosin, a glycosyl erythritol type biosurfactant produced by Pseudozyma flocculosa, which causes bursting of conidia of Erysiphe graminis f. sp. tritici (Hajlaouia and Belangera 1993; Hammami et al. 2011). However, to date, no such inhibitory roles of MELs against initial fungal behaviours have been elucidated, although several studies demonstrating the direct antimicrobial activities of compounds against several micro-organisms, such as Gram-positive bacteria (Kitamoto et al. 1993) and phytopathogenic fungi (Ishii et al. 2011), have been reported. The first objective of this study was to verify the ability of MELs to decrease the hydrophobicity of solid surfaces by comparing them to several commercial synthetic surfactants currently in use. Among the members of the MEL 216
family, we focused on MEL-A, MEL-B, MEL-C and isoMEL-B, which are all di-acylated MELs, because their physiochemical properties have been well characterized, and information about processes relating to their massproduction are available (Arutchelvi et al. 2008; Fukuoka et al. 2008). IsoMEL-B is now commercially available from TOYOBO Co. Ltd. (Osaka, Japan). After demonstrating that MELs changed the hydrophobicity of the surfaces, we verified the effects of MEL treatments on the early infection behaviours of representative phytopathogenic fungal conidia, as well as their suppressive activities against disease occurrences on host leaves caused by the fungal conidia. As model plants, we used the leaves of two Gramineae plants (i.e. wheat, rice), the surfaces of which are highly hydrophobic (Watanabe and Yamaguchi 1991), and the leaves of two non-Gramineae plants (i.e. strawberry and mulberry), which are presumably more hydrophilic than those of the former plant family (Watanabe and Yamaguchi 1991; Fukuoka et al. 2015). Materials and methods Materials The surfactants used in this study are members of the MEL family, MEL-A, MEL-B, MEL-C and isoMEL-B, which are produced by various species of Pseudozyma yeast strains (Morita et al. 2007; Fukuoka et al. 2008). Each MEL was isolated and purified from culture filtrates of the Pseudozyma strains, as described previously (Kitamoto et al. 1990a; Morita et al. 2007; Fukuoka et al. 2008). As synthesized surfactants, polyoxyethylene (20) sorbitan monolaurate (Tween 20) (Wako chemical, Tokyo, Japan) and polyoxyethylene (10) lauryl ether (Brij 35) (Wako chemical) were also used. The surfactants were individually adjusted to 01% (w/w) concentrations in sterilized distilled water (SDW), and were used for the following experiments after standing for at least 24 h. A MEL mixture (01%) extracted from the culture broth of Pseudozyma antarctica T-34 that contained MEL-A, MELB and MEL-C (mixing ratio, 58 : 25 : 10) (Kitamoto et al. 1990b) was alternatively used for each compound in a disease inhibition assay against Magnaporthe grisea, because preliminary experiments using each MEL compound did not inhibited the disease (data not shown). All of the reagents and solvents were commercially available and used as received. Measurement of contact angles of water droplets on plastic surfaces pretreated with surfactants The droplet contact angles (h) of water droplets on abiotic surfaces were evaluated based on Young’s equation
Journal of Applied Microbiology 119, 215--224 © 2015 The Society for Applied Microbiology
S. Yoshida et al.
(Adamson and Gast 1997); for droplets placed on a surface, smaller values of h indicate stronger attraction to the solid surface. As the abiotic surface, small segments (2–3 cm2) of plastic film (Gelbond: Takara, Otsu, Japan) were used for the measurements. The film was immersed in a surfactant solution or in SDW as a control. After air-drying, an SDW droplet (10 ll) was placed onto the hydrophobic side of the film segment, and the h value of the droplet at 100 s after placement was measured using Drop Master DM500 (Kyowa Interface Science Co., LTD, Niiza, Japan). Measurements were independently repeated in triplicate, and the obtained data were averaged and then subjected to ANOVA followed by Scheffe’s test using the KALEIDAGRAPH 4.1 J software (HULINKS Inc., Tokyo, Japan). Fungal isolates Wheat powdery mildew fungus, Blumeria graminis f. sp. tritici strain T-10 (hereafter Bg), mulberry anthracnose fungus, Colletotrichum dematium strain S9733 (hereafter Cd), strawberry anthracnose fungus, Glomerella cingulata strain S0709 (hereafter Gc) and rice blast fungus, M. grisea strain Kyu89–246 (hereafter Mg), all of which were preserved in our laboratory, were used in this study. Preparation of fungal inocula Conidia of the obligate parasitic fungus, Bg, were prepared according to the shaking method (Moseman et al. 1965). Briefly, potted wheat seedlings (cultivar (cv.) Ayahikari) grown in a glass house and artificially infested with Bg were periodically maintained by shaking off the conidia, thereby presenting the symptom to new healthy seedlings; the diseased seedlings harbouring fresh symptoms and conidia on the plant surface were used as the inoculum. The conidial suspension of Cd was prepared according to the method previously reported by Yoshida et al. (2000). Briefly, conidia of Cd harvested from 10day-old mulberry leaf decoction sucrose agar (MSA: dried mulberry leaf, 50 g; sucrose, 30 g; agar, 27 g; distilled water, 15 l) were washed twice in SDW by centrifugation (6000 g for 5 min), and then adjusted to a concentration of 5 9 105 conidia ml 1 using a hemocytometer. To prepare the conidial suspension of Gc, the fungal strain was shake-cultured (100 rev min 1) in a 200 ml Erlenmeyer flask containing 30 ml potato dextrose broth (Difco, Detroit, MI) at 25°C for 5 days. Conidia in the culture were harvested by pouring the incubation mixture through cotton mesh, followed by centrifugation (6000 g for 5 min). The harvested conidia were washed twice by centrifugation and adjusted to a concentration of 5 9 105 conidia ml 1, as described above. Conidia of Mg
MELs on hydrophobicity and fungal infection
were produced on the surface of colonies of oatmeal agar (Difco) plates and incubated at 25°C under 12 h black light (BLB) light:12 h dark conditions, harvested in SDW with an artist’s brush, and then adjusted to a concentration of 5 9 105 conidia ml 1, as described above. Observation of conidial germination and appressorial formation on hydrophobic plastic surfaces pretreated with surfactants To observe the effects of surface hydrophobicity attributed to surfactants against conidial germination, lengths of germ tubes and appressorial formation, 10 ll of each surfactant solution were placed on the hydrophobic surface of 10 Gelbond film segments (approx. 2 cm2) and then air-dried. Untreated segments were also prepared as a control. Subsequently, aliquots (5 ll) of the conidial suspensions of each fungus, with the exception of Bg, prepared as described above, were placed onto the film segments treated with surfactants. To analyze Bg, conidia appearing on diseased wheat seedlings were sprinkled onto the surface of each surfactant-treated Gelbond segment by the shaking method (Moseman et al. 1965), and approx. 90 conidia mm 2 were attached to the surface. The segments were then incubated in a moist plastic chamber at 25°C in the dark for 1 day, and 2–3 ll lactophenol cotton blue reagent (phenol, 10 g; lactic acid, 10 g; glycerol, 20 g; distilled water, 10 g; cotton blue, 0025 g) were added to each droplet on the segments to stain and fix fungal tissues, after which the droplets were mounted with cover slips. Fungal morphologies (ratios of conidial germination, lengths of germ tubes, ratios of appressorial formation) on each segment were observed using a light microscope (Eclipse 80i; Nikon, Tokyo, Japan). The ratio of conidial germination was determined by observing 50 randomly chosen conidia of each fungal strain. For evaluation of germ tubes lengths, the degrees of the lengths of germinated conidia were determined based on the length relative to that of the main axis of the conidia with the following index values: 1, ≤1-fold; 2, >1-fold to 2-fold; 3, >2-fold to 4-fold; 4, >4-fold. The ratio of appressorial formation was evaluated by observing the formation of typical pigmented appressoria at the tips of germ tubes of the germinated conidia of each fungal strain. These observations were carried out in triplicate independently, and the averaged data were subjected to ANOVA followed by Dunnett’s test using the KALEIDAGRAPH 4.1 J software. Disease suppression assay For each surfactant solution, after treatment on the surface of leaves, suppressive activity against symptom
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Results Contact angles of water droplets on plastic surfaces pretreated with surfactants The initial averaged h value of SDW droplets on the SDW-treated plastic film was 813° (data not shown), and the value at 100 s after placement was 763° (Fig. 1). Under such hydrophobic surface conditions, h values of SDW droplets on surfactant solution-treated surfaces were verified to decrease at 100 s after dropping (Fig. 1). The droplet angles at 100 s on surfaces treated with MEL-A and isoMEL-B were below 55 and 70°, respectively, and their angles were not significantly different
Contact angle of droplet (θ )
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index values 0–6, representing the ratio of lesion area (0, no lesion; 1, trace appearance; 2, ≤5%; 3, >5 to 10%; 4, >10 to 25%; 5, >25 to 50%; 6, >50%), on three randomly chosen mature leaves per seedling. Disease suppression in each inoculation test was evaluated based on the averaged values among the triplicated data, and the averages were subjected to ANOVA followed by Dunnett’s test as described above.
M
development of powdery mildew on wheat leaves from Bg, anthracnose of mulberry and strawberry from Cd and Gc, respectively, and rice blast from Mg, was evaluated. For the assay against powdery mildew of wheat, 10 cm long primary leaves of potted wheat seedlings (cv. Ayahikari) grown in the glass house as described above were cut out. The leaf segments were then immersed in each surfactant solution in a test tube. After the leaves were air-dried, conidia of Bg on diseased wheat leaves were sprinkled onto the surfaces of the treated leaves by the shaking method, as described above. The inoculated leaves were incubated in moist chambers for 7 days at 20°C under 12 h light: 12 h dark conditions. The degree of necrotic symptom appearance was evaluated based on lesion index values 0–4, representing the ratio of the lesion area on each leaf (0, no lesion; 1, ≤10%; 2, >10 to 25%; 3, >25 to 75%; 4, >75%). Five leaf replicates were used for each surfactant treatment. In the suppressive assay against mulberry anthracnose from Cd, small segments (approx. 15 cm2) of mature healthy mulberry leaves detached from a mulberry tree (unknown cultivar) planted on the grounds of National Institute for AgroEnvironmental Sciences, Tsukuba, were prepared. Aliquots (10 ll) of each surfactant solution were placed on the adaxial surfaces of individual segments. After the solutions were air-dried, 5 ll of conidial suspension (105 conidia ml 1), prepared as described above, were placed onto the surfaces of the treated segments. Suppressive activity resulting from treatment was evaluated based on the appearance and diameter (mm) of the round and brown lesions on leaf segments after 7 days of incubation in a moist chamber at 25°C in the dark. Seven replicate leaf segments were used for each surfactant treatment. The experiment against strawberry anthracnose caused by Gc was performed using segments (approx. 15 cm2) of strawberry leaves (cv. Nyohou) dissected from potted nurseries grown in a glass house. Treatments of surfactant solutions, inoculation of the conidial suspension (105 conidia ml 1), and evaluation of suppressive activity were performed in the same manner as the experimental treatment of mulberry leaves against Cd. For suppressive activity against rice blast disease caused by Mg, 9–10 rice seedlings (cv. Koshihikari) at the fourth–fifth leaf stage in a plastic nursery box (15 9 6 9 10 cm) grown in a glass house were used. A MEL mixture (20 ml; a substitute for MEL-A, MEL-B and MEL-C), Tween 20 and Brij 35, were sprayed onto the seedlings in the nursery box, and then air-died. Then, 20 ml of conidial suspension of Mg (5 9 105 conidia ml 1) was similarly sprayed onto the seedlings. The inoculated seedlings were incubated in a moist chamber for 2 days at 25°C, and then kept in a glass house for 5 days at 25°C. Subsequently, the degree of symptom appearance was evaluated based on lesion
Surface treatment Figure 1 Contact angles of sterilized distilled water droplets on plastic (Gelbond) surfaces pretreated with surfactant solutions at 100 s after placement. The data represent averages from three independent experiments, and bars indicate the standard error of the mean. ND indicates that measurements of angles were not determined. The same letter above multiple columns indicates that the values were not significantly different according to Scheffe’s test (P < 005).
Journal of Applied Microbiology 119, 215--224 © 2015 The Society for Applied Microbiology
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MELs on hydrophobicity and fungal infection
from those on the Tween 20-treated surfaces (h = 68), whereas droplets on the Brij 35 (h = 93) and water-treated surfaces exhibited significantly (P < 005) larger angles. Because droplets on the MEL-B and MEL-C-treated surfaces immediately spread over the limit of detection, their h values at 100 s were not determined. Effects of surfactant treatments on conidial germination and lengths of germ tubes on hydrophobic plastic surfaces Figure 2 shows the ratios of conidial germination of the examined fungal isolates on the surfactant-treated Gelbond film segments. Against Bg conidial germination, treatments of MEL-A and isoMEL-B significantly (P < 005) suppressed germination, with a germination ratio of
